CN103534392A

CN103534392A - Surface, anchored FC-bait antibody display system

Info

Publication number: CN103534392A
Application number: CN201180066437.XA
Authority: CN
Inventors: D.查; H.H.沙希恩
Original assignee: Schering Corp
Current assignee: Merck Sharp and Dohme LLC
Priority date: 2010-12-01
Filing date: 2011-11-29
Publication date: 2014-01-22
Also published as: WO2012074948A2; US20140186334A1; US9365846B2; EP2646606A2; CN107459577A; EP2646606B1; CN110042066A; WO2012074948A3; EP2646606A4

Abstract

The present invention provides, in part, an antibody display system that simultaneously uses a secretion and a display mode. A bait complexed with a monovalent antibody fragment can be expressed on the surface of the host cell wherein the fragment may be assayed for antigen binding while full antibody is simultaneously secreted from the host cell. Methods of using the system for identifying antibodies that bind specifically to an antigen of interest are also provided. Polypeptides, polynucleotides and host cells useful for making the antibody display system are also provided along with methods of use thereof.

Description

The FC-bait antibody display systems of surface anchoring

The application requires the interests of the U.S. Provisional Patent Application submitted on December 1st, 2011 number 61/458,771, and described U.S. Provisional Patent Application integral body is incorporated herein by reference.

Invention field

The field of the invention relates to antibody display systems and the using method for the identification of the antibody of being combined with antigen-specific.

background of invention

For building with the technology in screening antibodies library, be phage display, target protein matter is expressed as the polypeptide fusions with bacteriophage coat protein matter thus, and subsequently by screening with fixing or the biotinylated ligand binding of solubility.

Yet phage display has several shortcomings.For example, protein and the cell surface proteins of some eucaryon secretions need posttranslational modification, for example glycosylation or extensively disulfide linkage isomerization, and it cannot obtain in bacterial cell.

Current yeast surface antibody display systems for example cold catching also has a plurality of shortcomings.In cold capture antibody display systems, at low temperature, from the antibody dispose procedure of host cell transhipment vesica, postpone, thereby make the antibody of secretion can be on cell surface with regard to antigen in conjunction with measuring.Cold catching method has the shortcoming of low signal-to-noise ratio, and for the evaluation that target antigen has specific antibody, depends on to a great extent the cellular expression levels of antibody.

Affinity matrix system for example by vitamin H by antibody and host cell surface coupling, they can be with regard to antigen in conjunction with measuring thus.Affinity matrix system demonstrates the high rate of the crossed contamination between antibody cloning.Antibody can become from host cell uncoupling, thereby and loses they and being connected of polynucleotide of its immunoglobulin chain of coding.

Full length antibody display systems by binding domain-immunoglobulin in conjunction with albumen for example albumin A full length antibody is tied on host cell surface, described immunoglobulin-binding proteins merges to cell surface anchorin.The polynucleotide that host cell contains encoding antibody immunoglobulin chain.Usually, the immunoglobulin-binding proteins that is combined in of antibody is expressed rear generation on cell surface.Therefore this system causes antibody and does not express some erroneous combination of the host cell of this antibody.

summary of the invention

The present invention partly provides the antibody display systems of the shortcoming without current available system.The present invention also allows antibody to show and the coupling of producing bacterial strain selection.By surface display, screen the bacterial strain of finding and can become production bacterial strain, preserve antibody sequence and integrity simultaneously.This method makes it possible to screening parameter, and for example antibody is folding and expression.

The invention provides and comprise following antibody display systems: (a) separated eukaryotic host cell (for example Pichia ( pichia) cell, for example pichia pastoris phaff ( pichia pastoris)); (b) comprise and for example merge, for example, for example, to the Fc immunoglobulin domains of surface anchoring polypeptide or its function fragment or the bait (polynucleotide that wherein cell comprises the bait of encoding) of its function fragment (comprising CH3, CH2-CH3 or VH-CH1 polypeptide) (people); (c) one or more polynucleotide of coding immunoglobulin light chain variable region; (d) one or more polynucleotide of coding immunoglobulin heavy chain variable region.Optionally, antibody display systems also comprises the non-whole antibody tying, and it comprises described immunoglobulin light and heavy chain; And/or the monovalent antibody fragments compound with the Fc part of bait.In one embodiment of the invention, different colony (for example immunoglobulin (Ig) library) in the heredity of described one or more polynucleotide of coding immunoglobulin light chain variable region from immunoglobulin light chain variable region; And/or, different colony (for example immunoglobulin (Ig) library) in the heredity of described one or more polynucleotide of the immunoglobulin heavy chain variable region of wherein encoding from immunoglobulin heavy chain variable region.In one embodiment of the invention, the polynucleotide that host cell comprises the bait of encoding, itself and adjustable promotor are (for example gUT1promotor, gADPHpromotor or pCK1promotor) operationally combination.

The present invention also provides separated bait polypeptide, for example comprise merge to the Fc immunoglobulin domains of surface anchoring polypeptide (for example SED1) or its function fragment or its function fragment (for example wherein Fc derived from IgG1, IgG2, IgG3 or IgG4 immunoglobulin (Ig); For example people, for example, comprise VH-CH1, CH2-CH3 or CH3 polypeptide).The encode polynucleotide of any separation of this type of polypeptide; The carrier that comprises polynucleotide forms part of the present invention with the separated host cell that comprises polynucleotide and carrier.Scope of the present invention comprises separated host cell (for example eukaryotic host cell, for example Pichia, for example pichia pastoris phaff), and it also comprises one or more polynucleotide (for example, from library) of the immunoglobulin light chain variable region of encoding; And/or one or more polynucleotide (for example, from library) of coding immunoglobulin heavy chain variable region.In one embodiment of the invention, host cell of the present invention comprises and is positioned at for example polypeptide on cytolemma of cell surface.

The present invention comprises separated host cell (Pichia for example, pichia pastoris phaff for example), its cell surface anchor (for example SED1) that comprises the part that is for example bait by it be positioned on host cell surface, the bait polypeptide compound with Fc/ Fab; Optionally wherein said Fc/ Fab is combined with antigen; Optionally comprise antibody or its Fab, the light and heavy chain immunoglobulin that it comprises Fc/ Fab; For example, wherein host cell comprises for example one or more polynucleotide of bait, light chain immunoglobulin (Ig) and/or heavy chain immunoglobulin of encoding.

The present invention also provides the composition that comprises host cell of the present invention (referring to for example above), and described composition also comprises the non-whole antibody tying, and it comprises described immunoglobulin light and heavy chain; And/or with the Fc/ Fab (for example monovalent antibody fragments) of the compound antibody of the Fc part of bait.In one embodiment of the invention, described whole antibody or Fc/ Fab and antigen are compound.

The invention provides the method for whether being combined with antigen-specific for measuring antibody or its Fab, it comprises makes antibody display systems contact with described antigen; Wherein said antibody display systems comprises: (a) separated eukaryotic host cell (for example Pichia, for example pichia pastoris phaff), its polynucleotide that comprise the light chain immunoglobulin of encoding (for example, from library); Polynucleotide (for example, from library) with coding heavy chain immunoglobulin; (b) comprise and for example merge, for example, for example, to the lip-deep surface anchoring polypeptide of described eukaryotic host cell or the Fc immunoglobulin domains of its function fragment (SED1) or the bait of its function fragment (people, comprises VH-CH1, CH2-CH3 or CH3 polypeptide); On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab (for example monovalent antibody fragments) heavy and light chain immunoglobulin compound; Whether and measuring described Fc/ Fab (for example monovalent antibody fragments) is combined with described antigen-specific; If wherein monovalent antibody fragments is combined with described antigen-specific, described TPPA is antigen described in specific binding.In one embodiment of the invention, the method also comprises separated one or more polynucleotide, and optionally measures nucleotide sequence.In one embodiment of the invention, the method also comprises the expression that suppresses described bait, measures subsequently the antibody of described evaluation or its Fab for the avidity of described antigen.In one embodiment of the invention, the method also comprises the recombinant expressed immunoglobulin chain by this polynucleotide encoding, the antibody or its Fab that comprise described immunoglobulin (Ig) with optionally separating, and the optional pharmaceutical preparation that comprises the described antibody of combination or its Fab and pharmaceutically acceptable carrier that produces.

The present invention also provides for the identification of following method:

(i) the antibody of being combined with antigen-specific or its Fab; Or (ii) encoding said antibody or the polynucleotide (for example, from library) of heavy chain immunoglobulin of fragment and/or the polynucleotide (for example, from library) of the light chain immunoglobulin of encoding said antibody or fragment; Described method comprises makes antibody display systems contact with described antigen, wherein said antibody display systems comprises: (a) separated eukaryotic host cell (Pichia for example, pichia pastoris phaff for example), the polynucleotide that it comprises the light chain immunoglobulin of encoding; Polynucleotide with coding heavy chain immunoglobulin; With

(b) comprise and for example merge, for example, to the Fc immunoglobulin domains of the lip-deep surface anchoring polypeptide of described eukaryotic host cell or its function fragment (SED1) or its function fragment (people; For example comprise VH-CH1, CH2-CH3 or CH3 polypeptide) bait; On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab (for example monovalent antibody fragments) heavy and light chain immunoglobulin compound; Whether and measuring described Fc/ Fab (for example monovalent antibody fragments) is combined with described antigen-specific; If wherein observe the described specific binding with described antigen, identify this antibody or fragment or polynucleotide.In one embodiment of the invention, the method also comprises separated one or more polynucleotide, and optionally measures nucleotide sequence.In one embodiment of the invention, the method also comprises the expression that suppresses described bait, measures subsequently the antibody of described evaluation or its Fab for the avidity of described antigen.In one embodiment of the invention, the method also comprises the recombinant expressed immunoglobulin chain by this polynucleotide encoding, the antibody or its Fab that comprise described immunoglobulin (Ig) with optionally separating, and the optional pharmaceutical preparation that comprises the described antibody of combination or its Fab and pharmaceutically acceptable carrier that produces.

The present invention also provides the method for the preparation of antibody display systems, and it comprises: (a) separated eukaryotic host cell (for example Pichia, for example pichia pastoris phaff); (b) comprise and for example merge, for example, to the people Fc immunoglobulin domains of surface anchoring polypeptide or its function fragment (SED1) or its function fragment (people; For example comprise VH-CH1, CH2-CH3 or CH3 polypeptide) bait; (c) one or more polynucleotide (for example, from library) of coding immunoglobulin light chain variable region; (d) one or more polynucleotide (for example, from library) of coding immunoglobulin heavy chain variable region; Described method comprises the polynucleotide of the described bait of coding, described one or more polynucleotide of coding immunoglobulin light chain variable region; Introduce in described eukaryotic host cell with described one or more polynucleotide of coding immunoglobulin heavy chain variable region.

The present invention also provides the method for the preparation of antibody or its Fab, and it comprises one or more polynucleotide of coding immunoglobulin light chain variable region; And/or one or more polynucleotide of coding immunoglobulin heavy chain variable region are introduced the separated eukaryotic host cell that comprises bait (Pichia for example, for example, pichia pastoris phaff), described bait comprises and for example merging, for example, to the people Fc immunoglobulin domains of surface anchoring polypeptide or its function fragment (SED1) or its function fragment (people; For example comprise VH-CH1, CH2-CH3 or CH3 polypeptide); With cultivate host cell expressing thus under the polynucleotide of coding immunoglobulin chain and the condition by described chain formation antibody or its Fab; Wherein said bait and adjustable promotor are (for example gUT1promotor, gADPHpromotor or pCK1promotor) operationally combination, and when described immunoglobulin chain is expressed, bait is expressed suppressed.

The present invention also comprises the method for the preparation of antibody or its Fab, and it is included in and allows under the light chain immunoglobulin of described antibody or fragment and condition that heavy chain immunoglobulin is expressed the eukaryotic host cell (for example pichia pastoris phaff) of culture of isolated in growth medium; Wherein said eukaryotic host cell comprises: (i) the polynucleotide of the described light chain immunoglobulin of encoding; (for example wherein said chain is by common polynucleotide or two polynucleotide encodings that separate with the polynucleotide of the described heavy chain immunoglobulin of encoding said antibody or fragment; And/or, wherein said described in one or two polynucleotide derive from library or derive from mono-clonal source); (ii) comprise and for example merge, for example, to the lip-deep surface anchoring polypeptide of described eukaryotic host cell or the Fc immunoglobulin domains of its function fragment or the bait of its function fragment (people, comprises VH-CH1, CH2-CH3 or CH3 polypeptide); On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab (for example monovalent antibody fragments) heavy and light chain immunoglobulin compound; And wherein the expression of bait is optionally repressed; Wherein said antibody or fragment are optionally secreted by described eukaryotic host cell; Described method optionally comprises from described eukaryotic host cell and separated described antibody or fragment substratum.

The present invention for example also provides, for measuring sugar (O-glycan and/or N-glycan, any in those is for example discussed herein) method to the effect of the antibody of being combined with antigen-specific or its Fab, it comprises makes antibody display systems be combined with described antigen; Wherein said antibody display systems comprises:

(a) separated eucaryon is controlled glycosylated host cell (for example pichia pastoris phaff), the polynucleotide that it comprises the light chain immunoglobulin of encoding; Polynucleotide with coding heavy chain immunoglobulin; (b) comprise surface anchoring polypeptide or the Fc immunoglobulin domains that comprises described sugar of its function fragment or the bait of its function fragment (for example people, for example, comprise VH-CH1, CH2-CH3 or CH3 polypeptide) merging on described host cell surface;

On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab heavy and light chain immunoglobulin compound; Wherein said heavy and/or light chain comprises described sugar;

Whether measure described Fc/ Fab is combined with described antigen-specific; The antibody that mensuration comprises described sugar or its Fab are for the binding affinity of antigen; The antibody being equal to for the avidity of antigen and other aspects that lack described sugar with relatively antibody or its Fab or its Fab are for the avidity of antigen; If wherein comprise the antibody of described sugar or the avidity of its Fab higher than the avidity that lacks this sugared antibody or its Fab, this sugar determination is for increasing the avidity for antigen, if and/or wherein comprise the antibody of described sugar or the avidity of its Fab lower than the avidity that lacks this sugared antibody or its Fab, this sugar determination is for reducing the avidity for antigen.

accompanying drawing summary

Fig. 1. antibody display systems of the present invention and using method thereof.The polynucleotide of encoding antibody and bait coexpression in pichia pastoris phaff.The polynucleotide of one or two antibody immunoglobulin chain of coding can or can be originated from mono-clonal from library.Pichia cell is expressed bait on cell surface, and some in this type of bait are by unit price polypeptide fragment (the comprising a heavy and light chain) combination of the antibody of expressing equally.The antibody of some expression is escaped bait combination, and is therefore soluble.Can confirm the expression of antibody on cell by facs analysis, and can measure the titre of cell antibody expression level.Close bait and express, or from cell, evict the polynucleotide of (or knocking out) coding bait from.Resulting cell is only expressed the polynucleotide of encoding antibody weight and light chain and is only produced solubility whole antibody.The cellular expression levels of antibody can be confirmed subsequently, and the mensuration of affinity of antibody can be carried out.

Fig. 2. the plasmid figure of pGLY9008.Merge to yeast saccharomyces cerevisiae ( saccharomyces cerevisiae) sED1homo sapiens ( homo sapiens) Fc is by pichia pastoris phaff aOX1promoters driven.

Fig. 3 (a & B). (a) measure the ELISA of κ light chain concentration for measuring the concentration of the antibody of being secreted by the bacterial strain of table 1 explanation.Swimming lane 1 is the ELISA standard of serial dilution; Swimming lane 2-3 contains the material producing by the bacterial strain in table 1, does not contain the Fc bait (SAFB) of surface anchoring; Swimming lane 5-10 contains identical bacterial strain and adds SAFB.Y8316 does not express antibody and is used as negative control.(b) supernatant liquor generating by the bacterial strain in 3a moves on albumin A post, with the antibody of catching secretion.The IgGs of wash-out moves on non-reduced SDS-PAGE.

Fig. 4 (a-c). figure has shown the FACS data of the different fluorescence intensities that confirmation is observed between a plurality of Pichia Pastoris strains.(a) express anti-HER2 and anti-PCSK9 and do not contain the parent strain of Fc-SED1 bait; (b) containing showing cell with the anti-Her2 that does not contain bait; (c) containing showing cell with the anti-PCSK9 that does not contain bait.

Fig. 5. show and anti-PCSK9 monovalent antibody fragments (H+L) or anti-Her2(H+L) the pichia pastoris phaff yeast strain YGLY21610 of the mark of the compound Fc-Sed1p of monovalent antibody fragments and the facs analysis of YGLY21614.For cell, the streptavidin of the anti-human Fc Alexa 488 of goat, biotinylated PCSK9 and APC 635 marks carries out double-tagging.Cell separately analyzed to (being respectively left and middle figure) and mixed (right figure) with 1:00 ratio.The point that represents YGLY21610 cell in right figure is in circle.

Fig. 6. express Fc-SED1 bait pichia pastoris phaff cell facs analysis and; (1) anti-PCSK9 antibody A X189 weighs and light chain (anti-PCSK9; AX189-Fc-Sed1p); Or (2) are from AX189 light chain and the heavy chain (BP550-Fc-Sed1p) in BP550 library; Or (3) are from AX189 heavy chain and the light chain (BP551-Fc-Sed1p) in BP551 library.Left figure shows the data relevant with the unfiled bacterial strain that contains library, and right figure shows the data relevant with the sorting cell that comprises library once or twice.The FACS data relevant with contrast AX189 express cell have also been shown.

Fig. 7. PCSK9 and the κ elisa assay in sorting storehouse taken turns in the BP550 of Pre-sorting and BP551 library and the 2nd.

Fig. 8. Fc-Sed1p shows the purposes that is found use in the novel different dimerization Fc fragment of using in dual specific and other application.In this method, lose itself and self and can be illustrated in (A) on cell surface with dimerization or with the Fc mutant of the ability of wild-type Fc different dimerization, and with H+L sudden change library coexpression, wherein Fab district remains unchanged, but CH2 and/or CH3 structural domain are (B) of sudden change.Use the surface display be combined with Fab, can use FACS separation for antigen in conjunction with positive cell (C).These cells will contain the novel Fc variant of the bait-Fc recovery dimerization to showing.Culture supernatant can be measured by SDS-PAGE, with the monomer secretion H+L(D that guarantees to contain novel Fc).This exercise by the novel different dimerization Fc that causes using same measured to implement follow-up transformation to or the evaluation (E) of mating partner.

detailed Description Of The Invention

The invention provides the method and system for antibody surface display, it is characterised in that displaying pattern and whole antibody secretion pattern simultaneously.Host cell is secreted whole antibody and on cell surface, is shown Fc/ Fab.This method utilization for example has cell surface proteins, as the Fc fusions (merging at N or C-terminal) of " bait ", described cell surface proteins is covalently coupled to cell surface (for example cell walls) or embeds in (partly or entirely) cytolemma (for example, as transmembrane protein), and with antibody (for example, from the monospecific antibody of Clone Origin or from the antibody in library) coexpression.The common dimerization of antibody molecule is to form in the endoplasmic reticulum of whole antibody molecule therein, and the Fc fusions " bait " of surface anchoring and monovalent antibody fragments different dimerization, be created in the mixture of showing on cell surface.Monovalent antibody fragments on cell surface can conjugated antigen.

Antibody forming system of the present invention can for example, adopt in any host cell (yeast, mammalian cell, bacterium), and wherein bait can be expressed and Fc/ Fab can be combined with bait on host cell surface.

The same dimerization that still occurs whole antibody, allows whole antibody molecule to be secreted in culture supernatants.The whole antibody of secretion for example can be for example for preclinical study after separation.

While needing, bait can be knocked or suddenly change or its expression can be closed, to prepare the bacterial strain that only produces whole antibody.

Molecular biology

According to the present invention, in art technology, there is conventional molecular biology, microbiology and the recombinant DNA technology that can adopt.This type of technology absolutely proves in the literature.Referring to for example, Sambrook, Fritsch & Maniatis, molecular Cloning:A Laboratory Manual, Second Edition(1989) and Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York(is in this article, and " Sambrook, waits people, 1989 "); dNA Cloning:A Practical Approach, I and II volume (D.N. Glover edits 1985); oligonucleotide Synthesis(M.J. Gait edits 1984); nucleic Acid Hybridization(B.D. Hames & S.J. Higgins edits (1985)); transcription And Translation(B.D. Hames & S.J. Higgins, edits (1984)); animal Cell Culture(R.I. Freshney, editor (1986)); immobilized Cells And Enzymes(IRL Press, (1986)); B. Perbal, a Practical Guide To Molecular Cloning(1984); F.M. Ausubel, waits people (editor), current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Library is generally the set of relevant but different polynucleotide, and described polynucleotide are generally in common carrier main chain.For example, the polynucleotide in common carrier main chain can be contained in light chain or heavy chain immunoglobulin library, and it is coded in light and/or heavy chain immunoglobulin different but relevant in its nucleotide sequence; For example, it is different in function that described immunoglobulin (Ig) for example forms in the ability of mixture at itself and other immunoglobulin (Ig)s in antibody display systems of the present invention, and in conjunction with specific antigen.

When the encoding sequence of sequence-directed RNA polymerase mediation is transcribed into RNA, preferred mRNA, described RNA subsequently can montage (if it contains intron) and is translated into the protein of being encoded by encoding sequence, encoding sequence " in cell transcribing and translating control sequence controls under ", " with in cell transcribe and translate in control sequence function in conjunction with " or " control sequence of transcribing and translate in cell is operationally combined ".Therefore, bait gene can for example adjustable promotor or constitutive promoter be operationally combined with promotor.

The polynucleotide of discussing herein form part of the present invention." polynucleotide ", " nucleic acid " or " nucleic acid molecule " comprise strand or double-stranded DNA and RNA.

In one embodiment of the invention, for example encoding the immunoglobulin chain of antibody display systems of the present invention or the polynucleotide of component (for example bait) can be by natural adjusting (express and control) sequence side joint, or can be combined with heterologous sequence, described heterologous sequence comprises promotor, internal ribosome entry site (IRES) and other ribosome bind site sequences, enhanser, response element, suppressor gene, signal sequence, polyadenylation sequence, intron, 5'-and 3'-non-coding region etc.

For example the encode immunoglobulin chain of antibody display systems of the present invention or polynucleotide of component can be operationally combined with promotor.In one embodiment of the invention, " promotor " or " promoter sequence " is can for example, in conjunction with the DNA regulatory region of transcribing of the RNA polymerase in cell (directly or by other promotor conjugated protein or material) and start code sequence.Promoter sequence generally be take transcription initiation site as boundary at its 3' end, and upstream (5' direction) extends, to be included in initial base or the element of transcribing required minimal number of any level.In promoter sequence, can find transcription initiation site (for example defining easily by mapping with s1 nuclease), and the protein-binding domains of responsible RNA polymerase combination (consensus sequence).Promotor can comprise enhanser and checks sequence or be combined with nucleic acid of the present invention with other expression control sequencs.The promotor that can express for controlling gene includes but not limited to cytomegalovirus (CMV) promotor (U.S.

Patent number

5, 385, 839 and 5, 168, 062), SV40 early promoter district (Benoist, Deng people, (1981) Nature 290:304-310), promotor (the Yamamoto containing in the 3' of Rous sarcoma virus long terminal repeat, Deng people, (1980) Cell 22:787-797), herpes thymidine kinase promoter (Wagner, Deng people, (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), adjusting sequence (the Brinster of metallothionein gene, Deng people, (1982) Nature 296:39-42), prokaryotic expression carrier for example β-lactamase promotor (Villa-Komaroff, waits people, (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731) or tacpromotor (DeBoer, waits people, (1983) Proc. Natl. Acad. Sci. USA 80:21-25), also referring to " Useful proteins from recombinant bacteria " in Scientific American(1980) 242:74-94, for example, with the promoter element from yeast or other fungies, gal 4promotor, aDC(alcoholdehydrogenase) promotor, pGK(phosphoglycerokinase) promotor or alkaline phosphatase promoter.

Term " carrier ", " cloning vector " and " expression vector " comprise vehicle (for example plasmid), DNA or RNA sequence can be introduced in host cell, to transform host and optionally promote the expression of institute's calling sequence and/or copy by it.In one embodiment of the invention, encoding the immunoglobulin chain of antibody display systems of the present invention or the polynucleotide of component (for example bait) can be in carrier.

The host cell that can use in composition of the present invention or method as discussed in this article, comprises that eukaryote is such as low etc. and higher eucaryotic cells and prokaryotic organism.Higher eucaryotic cells comprise Mammals, insect (for example the greedy noctuid in meadow ( spodoptera frugiperda) cell) and vegetable cell (for example protalixcell).In one embodiment of the invention, host cell is for example yeast or filamentous fungal cells of lower eukaryotes, its be for example selected from any Pichia cell, pichia pastoris phaff, Finland's pichia spp ( pichia flnlandica), happiness trehalose pichia spp ( pichia trehalophila), pichia koclamae, film mould pichia ( pichia membranaefaciens), small pichia spp ( pichia minuta) ( ogataea minuta, pichia lindneri), pichia opuntiae, pichia thermotolerans, willow pichia spp ( pichia salictaria), pichia guercuum, outstanding general pichia spp ( pichia pijperi), pichia stipitis ( pichia stiptis), pichia methanolica ( pichia methanolica), Pichia, yeast saccharomyces cerevisiae, Saccharomycodes ( saccharomyces), multiple-shaped nuohan inferior yeast ( hans β nula polymorpha), genus kluyveromyces ( kluyveromyces), Kluyveromyces lactis ( kluyveromyces lactis), Candida albicans ( candida albicans), Aspergillus nidulans ( aspergillus nidulans), aspergillus niger ( aspergillus niger), aspergillus oryzae ( aspergillus oryzae), Trichodermareesei ( trichoderma reesei), chrysosporium lucknowense, Fusarium ( fusari υ m), Fusarium graminearum ( fusa um gramineum), fusarium venenatum, and Neurospora crassa ( neuraspora crassa).High eukaryotic host cell comprises mammalian host cell for example Chinese hamster ovary (CHO) cell, bhk cell or NSO cell.Prokaryotic host cell can be bacterial cell for example, for example Escherichia ( escherichia), enterobacter ( enterobacter), Azotobacter ( azotobacter), erwinia ( erwinia), bacillus ( bacillus), Rhodopseudomonas ( pseudomonas), Klebsiella ( klebsiella), proteus ( proteus), salmonella ( salmonella), serratia ( serratia), Shigella ( shigella), rhizobium ( rhizobia), Vitreoscilla ( vitreoscilla) and paracoccus ( paracoccus).Intestinal bacteria ( e. coli) host cell comprise DHB4, BL21(its at the people such as Lon(Phillips (1984) J. Bacteriol. 159:283) and OmpT proteolytic enzyme in defect), HB101, BL21 DE3, intestinal bacteria AD494, intestinal bacteria W3110(ATCC 27,325), intestinal bacteria 294(ATCC 31,446), intestinal bacteria B and intestinal bacteria X1776(ATCC 31,537).Other bacterial strains comprise that it is people (1992) Science 258,987 such as intestinal bacteria B834(Leahy of methionine(Met) defect); Other bacterial strains comprise BLR bacterial strain, and K-12 bacterial strain HMS174 and NovaBlue, and it is the recA derivative (these bacterial strains can derive from Novagen) that improves plasmid monomer yield and can help the stable target plasmid that contains tumor-necrosis factor glycoproteins.Also referring to U.S. Patent number 4,952,496,5,693,489 and 5,869,320, and Davanloo, P., waits people, (1984) Proc. Natl. Acad. Sci. USA 81,2035-2039; Studier, F. W., waits people, (1986) J. Mol. Biol. 189:113-130; Rosenberg, A. H., waits people, (1987) Gene 56:125-135; And Dunn, J. J., waits people, (1988) Gene 68:259.Prokaryotic cell prokaryocyte also can be for example allowing polypeptide for example under the recombinant expressed condition of immunoglobulin polypeptides and/or bait, to cultivate in substratum.These class methods that comprise this genoid and protein and host cell are parts of the present invention.Prokaryotic host cell also can be as the host cell in antibody display systems of the present invention, as discussed in this article.

As used herein, term " N-glycan " and " sugared shape " are used interchangeably and refer to N connection oligosaccharides, for example, by l-asparagine-N-acetyl-glucosamine, connect asparagine residue that is attached to polypeptide.N connection glycoprotein contains the N-acetyl-glucosamine residue being connected with the amide nitrogen of asparagine residue in protein.The sugar of preponderating of finding on glycoprotein is glucose, semi-lactosi, seminose, Fucose, N-acetylgalactosamine (Gal Ν Ac), N-acetyl-glucosamine (Glc Ν Ac) and sialic acid (for example N-acetyl-neuraminate (NANA)).

N-glycan has Man ₃glc Ν Ac ₂(" Man " refers to seminose; " Glc " refers to glucose; And " NAc " refers to N-ethanoyl; GIcNAc refers to N-acetyl-glucosamine) common pentose core.N-glycan for example, with regard to branch's (feeler) number that comprises periphery sugar (Glc Ν Ac, semi-lactosi, Fucose and sialic acid) difference, and described periphery sugar is attached to Man ₃glc Ν Ac ₂(" Man ₃") core texture, it is also referred to as " three seminose cores ", " pentose core " or " few seminose core ".N-glycan for example, according to its branch's moiety classify (high mannose, complexity or hybridization)." high mannose " type N-glycan has five or mannan residue more." complexity " type N-glycan generally has at least one the Glc Ν Ac that is attached to 1,3 seminose arm and at least one the Glc Ν Ac that is attached to 1, the 6 seminose arm of " three seminoses " core.Complicated N-glycan can also have semi-lactosi (" Gal ") or N-acetylgalactosamine (" Gal Ν Ac ") residue, and it optionally for example, is modified by sialic acid or derivative (" NANA " or " NeuAc ", wherein " Neu " refers to that neuraminic acid and " Ac " refer to ethanoyl).Complicated N-glycan also can have displacement in the chain that comprises " bisection " Glc Ν Ac and core Fucose (" Fuc ").Complicated N-glycan also can have a plurality of feelers in " three seminose cores ", is commonly called " many feelers glycan "." hybridization " N-glycan has zero at least one Glc Ν Ac on 1,3 seminose arm end of three seminose cores and 1, the 6 seminose arm in three seminose cores or a plurality of seminoses.A plurality of N-glycan are also referred to as " sugared shape "." PNG enzyme " or " glycanase " or " Polyglucosidase " refer to Peptide N-glycosidase F(EC 3.2.2.18).

In one embodiment of the invention, the O-glycosylation of the glycoprotein in " eukaryotic host cell " is in check.Scope of the present invention comprises that wherein O-glycosylation is the eukaryotic host cell (for example pichia pastoris phaff) of in check separation, and the antibody display systems that comprises this type of eukaryotic host cell and using method (as discussed in this article) thereof.For example, wherein O-glycan occupies with seminose chain length and reduces.At the low eukaryotic host cell that waits for example in yeast, can be by the encode gene (Dol-PMan: protein (Ser/Thr) mannose transferase gene) (PMTs) or by the host that grows in the substratum containing one or more Pmtp inhibitor control O-glycosylation of one or more protein O-mannose transferase of disappearance.Therefore, the present invention includes separated eukaryotic host cell, antibody display systems and the using method (as discussed in this article) thereof of the disappearance of one or more genes that for example comprise the PMTs that encodes, and/or for example, wherein host cell can be cultivated in the substratum that comprises one or more Pmtp inhibitor.Pmtp inhibitor comprises but is not limited to benzal thiazole diketone.The example of benzal thiazole diketone is 5-[[3, two (phenyl methoxyl group) phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; 5-[[3-(1-25 phenyl ethoxy)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; With 5-[[3-(1-phenyl-2-hydroxyl) oxyethyl group)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo-3-thiazolidine acetate.

In one embodiment of the invention, " eukaryotic host cell " comprises coding for alpha-1, the nucleic acid of 2 mannosidases, and it has and instructs its signal peptide for secreting.For example, in one embodiment of the invention, by transform host cell, be external source α-1 of expressing the best pH with 5.1-8.0, preferably 5.9-7.5,2 mannosidases.In one embodiment of the invention, by endoplasmic reticulum or the golgi body of exogenous enzyme target host cell, it revises for example Man of N-glycan therein ₈glcNAc ₂, to obtain Man _sglcNAc ₂.Referring to U.S. Patent number 7,029,872.

Scope of the present invention comprises this type of separated eukaryotic host cell (for example pichia pastoris phaff), and the antibody display systems that comprises this type of eukaryotic host cell and using method (as discussed in this article) thereof.

In one embodiment of the invention, " eukaryotic host cell " is the low eukaryotic cell (for example yeast, for example pichia pastoris phaff) that waits, for example, by (lacking or destroy one or more β-mannose transferase genes bMTl, bMT2, bMT3with bMT4) (referring to, U.S.'s publication application number 2006/0211085), or using RNA interfering, sense-rna etc. to cancel the translation of the RNAs of the one or more β-mannose transferases of coding, the described low eukaryotic cell that waits has the glycoprotein of alpha-Mannosidase resistance N-glycan with elimination through genetic modification.Scope of the present invention comprises this type of separated eukaryotic host cell (for example pichia pastoris phaff), and the antibody display systems that comprises this type of eukaryotic host cell and using method (as discussed in this article) thereof.

" eukaryotic host cell " also comprises the low eukaryotic cell (for example yeast and filamentous fungus, for example pichia pastoris phaff) of Denging, for example, by lacking or destroy one or two phosphomannose based transferase gene pNO1with mNN4B(referring to for example U.S. Patent number 7,198,921 and 7,259,007), it can comprise disappearance or destroy mNN4Agene or use RNA interfering, sense-rna etc. are cancelled the translation of the RNAs of the one or more phosphomannose based transferases of coding, and the described low eukaryotic cell that waits is the glycoprotein that elimination has phosphomannose residue through genetic modification.In one embodiment of the invention, " eukaryotic host cell " is genetically modified is selected from the glycoprotein of following N-glycan for producing to have to preponderate: complicated N-glycan, hybridization N-glycan and high mannose N-glycan, wherein in one embodiment of the invention, complicated N-glycan is selected from Man ₃glcNAc ₂, GlcNAC _(l-4)man ₃glcNAc ₂, NANA _(1-4)glcNAc _(l-4)man ₃glcNAc ₂and NANA _(l-4)gal _(1-4)man ₃glcNAc ₂; In one embodiment of the invention, hybridization N-glycan is selected from Man ₅glcNAc ₂, GlcNAcMan ₅glcNAc ₂, GalGlcNAcMan ₅glcNAc ₂and NANAGalGlcNAcMan ₅glcNAc ₂; And in one embodiment of the invention, high mannose N-glycan is selected from Man ₆glcNAc ₂, Man ₇glcNAc ₂, Mang ₈1cNAc ₂and Man ₉glcNAc ₂.Scope of the present invention comprises this type of separated eukaryotic host cell (for example pichia pastoris phaff), and the antibody display systems that comprises this type of eukaryotic host cell and using method (as discussed in this article) thereof.

As used herein, when it relates to the shortage of specific saccharide residue such as Fucose or semi-lactosi etc. on glycoprotein, term " is substantially free of " and is used to indicate glycoprotein compositions and substantially lacks the N-glycan that contains this type of residue.Expression with regard to purity, the amount that is substantially free of the N-glycan structures that means to contain this type of saccharide residue is no more than 10%, and preferably lower than 5%, more preferably less than 1%, most preferably lower than 0.5%, wherein per-cent is by weight or by mole% meter.

As used herein, when on N-glycan structures, do not exist can detection limit this type of saccharide residue time, the specific saccharide residue of glycoprotein compositions " shortage " is Fucose or semi-lactosi for example.For example, in a preferred embodiment of the invention, as discussed in this article, glycoprotein compositions produces by lower eukaryotes, and by " shortage Fucose ", because these biological cells do not have, does not produce the fucosylated required enzyme of N-glycan structures.Therefore, term " is substantially free of Fucose " and comprises term " shortage Fucose ".Yet, even if composition once contained fucosylated N-glycan structures as mentioned above, or containing fucosylated N-glycan structures limited but can detection limit, composition also can " be substantially free of Fucose ".

For example, for example as discussed in this article, the host cell of under specific circumstances, introducing, eliminate or be modified at the saccharide residue on the immunoglobulin (Ig) of expressing in host cell can be called as " controlling glycosylated host cell " in this article.

Fluorescence-activated cell sorting (FACS) is the flow cytometry of specialized types.It provides specific light scattering and fluorescent characteristics based on each cell, for the heterogeneous mixture of biomass cells is sorted into the method in two or more containers, and next cell.During cell sorting process, cell suspension drags at liquid stream center narrow, rapid flow.Flow and so arrange, thereby make at iuntercellular, to exist separated with respect to its diameter.Vibration mechanism causes that stream of cells fragments into indivedual droplets.Adjust like this this system, make to exist the low probability that surpasses a cell/droplet.Just in time, before stream fragments into droplet, flow through fluorescence measurement station, measure therein the object fluorescent characteristics of each cell.The charging ring stream that is just in time placed in one is fragmented on the point of droplet.Based on previously immediately fluorescence intensity measurement that electric charge is placed in to ring is upper, and when droplet is during from stream interruption, opposite charges is held back on droplet.Charged droplet falls subsequently through electrostatic deflection system, and its electric charge based on droplet redirect to it in container.In some systems, electric charge is directly applied to stream, and the droplet coming off retains and the electric charge that flows identical sign.After droplet comes off, stream is got back to neutrality subsequently.The present invention comprises for example method of antibody display systems of the present invention as discussed in this article of use, wherein based on cell whether by detectable fluorescent mark carry out mark (for example wherein antigen self or two anti-be mark), by FACS sorting, from unconjugated cell or the cell without the combination of enough levels, sub-elect and be combined the eukaryotic host cell of (by Fc/ Fab) with object antigen.The host cell of the mark of this type of sorting is also part of the present invention with the composition of the host cell of the mark that comprises this type of sorting.

Adjustable promotor is the promotor that its expression can be induced or suppress.Embodiment of the present invention comprise the antibody display systems that wherein expression of bait is controlled by adjustable promotor, with and using method as discussed in this article.With adjustable promotor operationally the polynucleotide of the coding bait of combination together with the separated eukaryotic host cell that comprises these polynucleotide, also form part of the present invention.The example of the adjustable promotor existing in yeast comprises gUT1promotor, gADPHpromotor and pCK1promotor.

In one embodiment of the invention, for example, for example, for example, by making cell be exposed to sense-rna or disturbing (Microrna (miRNA) or siRNA (siRNA)) to suppress the expression of polynucleotide (bait) in eukaryotic host cell (bait) by RNA.Embodiment of the present invention comprise the method for using antibody display systems (for example as discussed in this article), and wherein the expression of bait is disturbed by RNA or sense-rna is inhibited.The eukaryotic host cell (for example as discussed in this article) that comprises bait and also comprise the separation of the present invention that suppresses antisense that bait expresses or rnai molecule is also part of the present invention.

Antibody

For example, in conjunction with antibody or its Fab identified, can be reformated into any suitable form with purposes of the present invention (purposes of antibody display systems of the present invention).For example,, for example, from using the CDRs of the whole antibody of antibody display systems separation can mix in different frameworks (people's framework), to generate different antibodies or its Fab that comprises CDRs separated from antibody display systems of the present invention.Method for generation of chimeric, humanization and people's antibody is well-known in the art.Referring to for example, award to the people's such as Queen U.S. Patent number 5,530,101, award to the people's such as Winter U.S. Patent number 5,225,539, award to the people's such as Boss U.S. Patent number 4,816,397.For reformatting antibody or its Fab or be that this area is conventional and well-known for these class methods that CDRs is repositioned onto to another from a framework.For example, the CDRs of antibody or Fab can be for generating monoclonal antibody, polyclonal antibody, bi-specific antibody, chimeric antibody, recombinant antibodies, antiidiotypic antibody, humanized antibody and bi-specific antibody; Or its Fab for example nano antibody (nanobodies), Fab, Fab', F(ab') ₂, Fv fragment; DsFv; (dsFv) ₂, ds double antibody; DsFv-dsFv '; Single-chain antibody molecule is sc-Fv, sc-Fv dimer (divalence double antibody) for example; With dual specific double antibody.

The tetramer that whole antibody comprises subunit.Each tetramer has two pairs of polypeptide chains that are equal to, and every pair has " gently " chain (LC) (approximately 25 kDa) and " weight " chain (HC) (about 50-70 kDa).The N-terminal of every chain partly comprises the main approximately 100-110 of antigen recognition or the variable region of amino acids more of being responsible for.The C-terminal part qualifying part of every chain is responsible for the constant domain of effector function.Constant domain type based in light chain, is categorized as κ or λ by light chain (LCs).Constant domain type based in heavy chain, heavy chain (HCs) is categorized as to γ, μ, α, δ or ε, and the isotype of antibody is defined as respectively to IgG(for example IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4), IgM, IgA(for example IgA1 or IgA2), IgD or IgE.

The present invention comprises the method for the preparation of antibody or its Fab, and it comprises one or more polynucleotide of coding immunoglobulin light chain variable region; And/or one or more polynucleotide of coding immunoglobulin heavy chain variable region are introduced the separated host cell that comprises bait (eukaryotic host cell for example, Pichia for example, for example, pichia pastoris phaff), described bait comprises and for example merging, to the people Fc immunoglobulin domains of surface anchoring polypeptide or its function fragment or its function fragment (people; For example comprise VH-CH1, CH2-CH3 or CH3 polypeptide); With cultivate host cell expressing thus under the polynucleotide of coding immunoglobulin chain and the condition by described chain formation antibody or its Fab.

In one embodiment of the invention, described bait is operationally combined with adjustable promotor, and when described immunoglobulin chain is expressed, bait is expressed suppressed.In one embodiment of the invention, with sense-rna or by RNA, disturb inhibition bait to express.

The present invention for example also provides by enzyme-linked immunosorbent assay (ELISA) for measuring the method for the quantity of antibody or its Fab.For example, in one embodiment of the invention, the method comprises cultivates the eukaryotic host cell comprise isolated polypeptide, and described isolated polypeptide comprises bait polypeptide and (for example merges, to the Fc immunoglobulin domains of surface anchoring polypeptide or its function fragment or its function fragment (people; For example comprise VH-CH1, CH2-CH3 or CH3 polypeptide)); Wherein host cell is secreted whole antibody or its Fab (optionally, antibody or fragment are separated from host cell and/or substratum); And by ELISA, measure the quantity of antibody or its Fab.In one embodiment of the invention, the expression of bait is suppressed before quantitatively, thereby host cell is only expressed and secrete whole antibody.Bait polynucleotide can be operationally combined with adjustable promotor, suppress so described adjustable promotor, to suppress bait, express.For example, in one embodiment of the invention, ELISA comprises antigen coated on solid substrate; Antibody or its Fab are combined with antigen; Two of detectable label is resisted is combined with antibody or fragment; And detecting two resists.In one embodiment of the invention, two anti-ly carry out mark with alkaline phosphatase or horseradish peroxidase.In one embodiment of the invention, by making the absorbancy of alkaline phosphatase (AP) or horseradish peroxidase (HRP) and Binding Capacity and measurement plate, (for example HRP substrate 3,3', 5,5'-tetramethyl benzidine (TMB); HRP substrate 3,3'-diaminobenzidine (DAB); Or HRP substrate 2'-azino-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) (ABTS); Or AP substrate p-nitrophenyl phosphoric acid ester) carry out certification mark.

The present invention also provides for measuring antibody or its Fab (its eukaryotic host cell by antibody display systems of the present invention is secreted) for the method for the avidity of antigen.For example, avidity can be measured by the affine ELISA of standard, Biacore analysis or competition assay.

Antibody display systems

The invention provides and comprise following antibody display systems, composition or test kit: (1) eukaryotic host cell and (2) are included in N or C-terminal (optionally by peptide linker for example GGG) and merge the people Fc for example to the Fc(of surperficial anchor, for example comprise VH-CH1, CH3 or CH2-CH3 polypeptide) bait, described bait optional with signal sequence (for example α mating factor signal sequence, for example, from yeast saccharomyces cerevisiae) connection; Described system can be for example for the evaluation of antibody.Therefore, in one embodiment of the invention, the host cell expression antibody in this system and/or one or more immunoglobulin chain of its Fc/ Fab (for example light and heavy chain, for example wherein one or more chain is originated from library).In one embodiment of the invention, the immunoglobulin chain of antibody and/or its Fc/ Fab comprises from that being equal to or different CH2-CH3 polypeptide of bait.

The Fc/ Fab (1) of antibody and the Fc of bait part (for example people Fc, for example, comprise VH-CH1, CH3 or CH2-CH3 polypeptide) is compound, and (2) when with host cell surface on bait compound tense, be combined with antigen.The example of Fc/ Fab is the unit price fragment (being monovalent antibody fragments) of whole antibody.In one embodiment of the invention, bait comprises CH2-CH3 polypeptide or its function fragment, and it is different from the CH2-CH3 of the Fc/ Fab of antibody on one or more residues.In this type of embodiment of the present invention, when the Fc/ of bait and antibody Fab in conjunction with time, form the Fc structural domain of different dimerization.

Half that " monovalent antibody fragments " comprises antibody, CDRs and the heavy chain of antibody (VH-CH1-CH2-CH3) light chain of antibody (VL-CL) combination that comprises three pairings, for example wherein CH1 and CL are by disulphide bridges combination, and described monovalent antibody fragments can detect ground conjugated antigen.

" bait " for example comprises at N-terminal or C-terminal and merges Fc structural domain to surperficial anchor (for example people, rat, rabbit, goat or mouse Fc, the any part of heavy chain (for example people, rat, rabbit, goat or mouse) for example, for example CH3 polypeptide, VH-CH1 polypeptide or CH2-CH3 polypeptide), described bait has the functional property described herein (for example, as below set forth) that causes bait to work in antibody display systems of the present invention.In one embodiment of the invention, Fc structural domain can suddenly change like this, to improve the ability that it works in antibody display systems of the present invention, for example can add or mobile halfcystine or other residues, when with human IgG Fc or Fc/ Fab compound tense, allow widely disulphide bridges to form.The Fc that is suitable for using in bait comprises Fc(, comprise CH1 and/or CH2 and/or CH3 structural domain) or its function fragment (for example, from IgG1, IgG2, IgG3 or IgG4 or its mutant), when merging to surface anchoring protein, described Fc or its function fragment can be for example and the lip-deep human IgG Fc of eukaryotic host cell or Fc/ Fab dimerization." fraction-crystalline " C-terminal district of the antibody that in one embodiment of the invention, term " Fc " refers to comprise CH2 and CH3 structural domain.In one embodiment of the invention, the dimerization between bait Fc and Fc/ Fab was occurring to cell surface by routes in cell, once wherein Fc and Fc/ Fab just keep combination on cell surface.Generally speaking, in the situation that not there is not Fc/ Fab, the same dimerization of bait; Thereby comprise two surperficial anchors and two Fc structural domains.In one embodiment of the invention, comprise the light and heavy chain of dimerization each other with the whole antibody of bait coexpression, to form monovalent antibody fragments, the Fc dimerization of described monovalent antibody fragments and bait.

Antigen can be any immunogenic molecules or material, for example polypeptide (for example oligopeptides), cytolemma, cell extract or full cell.Polypeptide antigen comprises for example following polypeptide: chemokine, cytokine (for example inflammatory cytokine or chemokine), acceptor, PCSK9, granulocyte-CSF; Coagulation factors is Factor IX, factors IX and human protein C for example; Soluble IgE receptor α chain; Urokinase; Quimotrase and urea trypsin inhibitor; Igf binding protein; Type-1 insulin like growth factor acceptor, vascular epidermis somatomedin, Urogastron; Somatotropin releasing factor; The TNFR related protein of GITR(glucocorticoid inducible), annexin V fusion rotein; IL-23p19, IL-23p40, IL-23R, IL12R-β 1, TNF α (tumor necrosis factor alpha), TGF β (tumor necrosis factor β), IL-10, IL-17, TSLP(thymic stromal lymphopoietin), angiostatin; VEGF-2; Marrow sample precursor cell supressor-1; Osteoprotegerin (OPG), the RANK(Nuclear factor kappa B receptor activation factor) or RANKL(receptor activator of the nuclear factor-κappaB ligand); In one embodiment of the invention, wherein any can be people.

" surperficial anchor " is when merging with Fc or its function fragment, is expressed and is positioned to any polypeptide of cell surface, and wherein Fc/ Fab can be compound with Fc or its function fragment.The example of cell surface anchor is protein, such as but not limited to SED-1, α-lectin, Cwpl, Cwp2, GasI, Yap3, FIoIp1 Crh2, Pirl, Pir4, Tipl, Wpi, Hpwpl, Als3 and Rbt5; For example, yeast saccharomyces cerevisiae CWP1, CWP2, SED1 or GAS1; Pichia pastoris phaff SP1 or GAS1; Or multiple-shaped nuohan inferior yeast TIP1.In one embodiment of the invention, surperficial anchor is fixed (GPI) protein of any glycosyl-phosphatidyl inositol anchor.The fragment that the function fragment of surface anchor comprises the full polypeptide of surperficial anchor, when merging to Fc or its function fragment, described fragment can form function bait; For example wherein when expressing as Fc fusions in eukaryotic host cell, described fragment is positioned on cell surface, and wherein Fc can for example, form mixture with Fc/ Fab (monovalent antibody fragments).

As discussed in this article, for the suitable eukaryotic host cell using at antibody display systems of the present invention, be Pichia cell, for example pichia pastoris phaff.

Scope of the present invention is included in the separated eukaryotic host cell (for example pichia pastoris phaff) that comprises bait (for example merging to people Fc structural domain or its function fragment of surperficial anchor or its function fragment at N-terminal or C-terminal) on cell surface, wherein for example for example pass through, at (between the CH2-CH3 polypeptide and Fc/ Fab in bait) combination, this bait and Fc/ Fab dimerization between bait Fc and the heavy chain of monovalent antibody fragments.The present invention also comprises such composition, and it comprises the eukaryotic host cell that comprises bait and antibody or its Fab and/or its Fc/ Fab for example secreted in liquid medium within.

The invention provides for example for the identification of following method: (i) the antibody of being combined with object antigen-specific or its Fc/ Fab, and/or (ii) encoding said antibody or the polynucleotide of heavy chain immunoglobulin of fragment and/or the polynucleotide of the light chain immunoglobulin of encoding said antibody or fragment.In one embodiment of the invention, the method comprises:

(a) for example, in separated eukaryotic host cell (pichia pastoris phaff), coexpression bait (for example comprises and comprising and the cell surface anchor CH3 that for example SED1 is connected, the polypeptide of VH-CH1 or CH2-CH3 polypeptide) and one or more heavy and light immunoglobulin chain (for example wherein one or more in this type of chain by the polynucleotide encoding of originating from library), thereby the Fc making at bait part (for example comprises VH-CH1, CH3 or CH2-CH3 polypeptide) and the Fc/ Fab (for example monovalent antibody fragments) that comprises immunoglobulin chain between mixture form and be positioned on cell surface, for example, wherein host cell transforms with one or more polynucleotide of coding bait and immunoglobulin chain,

(b) eukaryotic host cell of the bait of Fc/ Fab (for example monovalent antibody fragments) dimerization of evaluation expression and antibody, it has detectable avidity (for example acceptable avidity) (for example, with the detectable combination of antigen) for antigen; For example wherein bait and the light and polynucleotide encoding of heavy chain immunoglobulin in eukaryotic host cell;

In one embodiment of the invention, whether analysis package, containing non-that tie, the whole antibody secreted that are for example equal to, with the light and heavy chain immunoglobulin variable domains of compound those of the bait immunoglobulin (Ig) of host cell expression (by), has detectable avidity to measure them.

In one embodiment of the invention, whole antibody is secreted in substratum from host cell.In one embodiment of the invention, whole antibody is separated from host cell.

In one embodiment of the invention, after step (b), suppress the expression of bait in host cell, but do not suppress the expression of whole antibody.In this embodiment of the present invention, host cell is only expressed whole antibody but is not expressed the polypeptide with any remarkable quantity.In one embodiment of the invention, once the expression of bait is suppressed, just analyze the whole antibody being produced by host cell, to measure it, whether there is detectable avidity (for example acceptable avidity); With

(c), if observe the detected combination of Fc/ Fab, for example wherein one or more polynucleotide of encoded light and/or heavy chain immunoglobulin are optionally separated from host cell, identify described antibody or Fab or polynucleotide.In one embodiment of the invention, measure the nucleotide sequence of polynucleotide.

In one embodiment of the invention, host cell population is expressed common bait and co-immunization sphaeroprotein heavy chain and for example from the multiple different light chain immunoglobulin (Ig)s in source, library, wherein can be identified and form indivedual light chain immunoglobulin (Ig)s of Fc/ Fab and tie and demonstrate the whole antibody that antigen is combined with bait.Similarly, in one embodiment of the invention, host cell population is expressed common bait and co-immunization sphaeroprotein light chain and for example from the multiple different heavy chains immunoglobulin (Ig) in source, library, wherein can be identified and form indivedual heavy chain immunoglobulins of Fc/ Fab and tie and demonstrate the whole antibody that antigen is combined with bait.

In one embodiment of the invention, having the host cell of the polynucleotide of the heavy and light chain immunoglobulin (Ig) of coding can also for example, for the non-antibody tying (whole antibody) or its Fab at cultivation expression-secretion.For example, in this embodiment of the present invention, the expression of bait is optionally repressed, thereby the bait not occurring with remarkable quantity is expressed.Host cell subsequently in substratum secretion thus, for example, cultivate under the non-antibody tying (whole antibody) or the condition of its Fab by host cell expression and secretion.The non-antibody tying or its Fab can be optionally from host cell with separated substratum.In one embodiment of the invention, immunoglobulin chain is transferred to host cell (for example lacking antibody display systems component) separately for recombinant expressed.

The invention provides for example for the identification of following method: (i) the antibody of being combined with object antigen-specific or its Fc/ Fab, described object antigen is included in second CH2-CH3 of first CH2-CH3 that is different from bait on one or more residues, or (ii) encoding said antibody or the polynucleotide of heavy chain immunoglobulin of fragment and/or the polynucleotide of the light chain immunoglobulin of encoding said antibody or fragment.In one embodiment of the invention, the method comprises:

(a) bait that for example, coexpression comprises first CH2-CH3 polypeptide in separated eukaryotic host cell (pichia pastoris phaff); For example, for example, together with the heavy immunoglobulin chain that comprises described second CH2-CH3 polypeptide (wherein said heavy immunoglobulin chain is originated from library) and light immunoglobulin chain (VL-CL), thereby make the mixture combination between first CH2-CH3 polypeptide of bait and second CH2-CH3 polypeptide of Fc/ Fab and be positioned on cell surface; For example, wherein host cell transforms with one or more polynucleotide of coding bait and immunoglobulin chain;

(b) identify the eukaryotic host cell of the bait of expression and Fc/ Fab dimerization, it has detectable avidity (for example acceptable avidity) for antigen; For example wherein bait and the light and polynucleotide encoding of heavy chain immunoglobulin in eukaryotic host cell; Optionally,

Antibody display systems of the present invention can the effect for the avidity of antigen for assessment of given glycosylation pattern antagonist or its Fab.Generally speaking, the ability of the Fc/ Fab of the glycosylation pattern that comprises change can just be assessed with the combination of antigen, can assess the avidity of whole antibody or its Fab after this.For example by use, work as for example host cell and/or by for example cultivating host as discussed in this article under the condition of modification of glycosylation patterns thus as discussed in this article of chain modification of glycosylation patterns while expressing, can be modified at the glycosylation pattern on the immunoglobulin chain of expressing in antibody display systems.For example, in one embodiment of the invention, the method comprises makes antibody display systems contact with described antigen; Wherein said antibody display systems comprises: (a) separated eucaryon is controlled glycosylated host cell, the polynucleotide that it comprises the light chain immunoglobulin of encoding; Polynucleotide with coding heavy chain immunoglobulin; (b) comprise the bait that comprises Fc immunoglobulin domains or its function fragment merging to the lip-deep surface anchoring polypeptide of described eukaryotic host cell or its function fragment; On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab heavy and light chain immunoglobulin compound; Wherein said heavy or light chain comprises described sugar; Whether measure described Fc/ Fab is combined with described antigen-specific; The antibody that mensuration comprises described sugar or its Fab are for the binding affinity of antigen; The antibody being equal to the avidity of relatively antibody or its Fab and other aspects that lack described sugar or the avidity of its Fab; If wherein comprise the antibody of described sugar or the avidity of its Fab higher than the avidity that lacks this sugared antibody or its Fab, this sugar determination is for increasing the avidity for antigen, if and/or wherein comprise the antibody of described sugar or the avidity of its Fab lower than the avidity that lacks this sugared antibody or its Fab, this sugar determination is for reducing the avidity for antigen.For example, the avidity that lacks sugared antibody or its Fab can be measured in a similar manner in antibody display systems of the present invention or avidity, or it can be by by currently known methods, for example ELISA, biacore measure or competition assay measurement avidity is directly measured.

Bait is expressed can be by any being inhibited in several acceptable methods.For example, the polynucleotide of coding bait (for example surperficial anchor and/or Fc) can be expressed by adjustable promotor, and the expression of described adjustable promotor can be inhibited in host cell.In one embodiment of the invention, bait is expressed by RNA interference, sense-rna, for example, from the sudden change of polynucleotide or the genetic mutation of removal or polynucleotide of the coding bait (surperficial anchor and/or Fc) of host cell, is inhibited, thereby makes not expressive function bait of host cell.

" acceptable avidity " refers to that antibody or Fab are for the avidity of antigen, and it is at least 10 ^-3m or larger avidity (more low number), for example 10 ^-3m, 10 ^-4m, 10 ^-5m, 10 ^-6m, 10 ^-7m, 10 ^-8m, 10 ^-9m, 10 ^-10m, 10 ^-11m or 10 ^-12m.

In one embodiment of the invention, the polynucleotide of the heavy and light chain of encoding antibody or Fc/ Fab (for example monovalent antibody fragments) in one or more polynucleotide library, the light and/or heavy chain immunoglobulin of described polynucleotide encoding (for example a library encode light chain and a library encoding heavy chain).In this embodiment, the Fc/ Fab of the surface anchoring on observing host cell surface is when object antigen is combined, and specific purpose immunoglobulin chain is different from other chains in library.

In one embodiment of the invention, the weight of expressing in antibody display systems or light chain immunoglobulin (Ig) are originated from library, and other immunoglobulin chains are the known strand of Clone Origin (from).In this embodiment of the present invention, as discussed in this article, antibody display systems can be for the identification of new library chain, and when with known chain coupling, described new library chain formation is expected antibody or its Fab.Alternately, antibody display systems can be for analysis package containing the antibody of two known immunoglobulin chains or the expression of its Fab with in conjunction with feature.

In one embodiment of the invention, by making cell incubation together with fluorescently-labeled antigen (biological example element mark), and by the cell of fluorescence-activated cell sorting (FACS) sorting/selections specific binding antigen, the cell that passes through the anchor Fc/ Fab that for example SED1 and cell tie that can detect that expression is combined with antigen.

In one embodiment of the invention, use the eukaryotic host cell of the bait of fluorescence-activated cell sorting (FACS) evaluation and sorting expression and Fc/ Fab dimerization.For example, in one embodiment of the invention, with fluorescent antigen or the cell of the bait of the anti-marker expression of fluorescence two of being combined with antigen equally and the Fc/ Fab dimerization on cell surface.Fluorescent mark is detected and with the signal that acts on sorting in FACS assorting room.The existence of the cell surface of bait/Fc/ Fab/antigenic compound is expressed in the cell indication of mark, and is collected in vessel, and not the cell harvesting of expression signal in vessel separately.Whether correspondingly, the present invention includes the method that comprises following step is combined with antigen-specific for measuring from antibody or its Fab in library:

(1) for example, by the following eukaryotic host cell (pichia pastoris phaff) that is transformed into the polynucleotide that comprise the bait of encoding:

(i) one or more immunoglobulin (Ig)s library, the polynucleotide that contain encoded light and heavy chain immunoglobulin;

(ii) one or more immunoglobulin (Ig)s library, the polynucleotide that contain coding light chain immunoglobulin (Ig) and monospecific polyclonal heavy chain immunoglobulin; Or

(iii) one or more immunoglobulin (Ig)s library, the polynucleotide that contain encoding heavy chain immunoglobulin (Ig) and monospecific polyclonal light chain immunoglobulin (Ig);

Wherein said chain can form antibody or its Fab;

(2) cell that in liquid medium within, growth transforms;

(3) allow bait to express on cell surface;

(4) with fluorescently-labeled antigen or with the antigenic mark cell of fluorescently-labeled two anti-bindings;

(5) use FACS sorting and separated fluorescently-labeled cell one to take turns;

(6) cell regrowth mark, sorting;

(7) allow bait at cells;

(8) with fluorescently-labeled antigen or with the antigenic mark cell of fluorescently-labeled two anti-bindings;

(9) use FACS sorting and separated fluorescently-labeled cell second to take turns;

(10) cell regrowth mark, sorting on solid medium, thus make indivedual cell clones grow into discontinuous cell bacterium colony;

(11) identify the bacterium colony for antigen with avidity;

(12) in liquid medium within, grow from the cell of identifying bacterium colony, and the separated non-supernatant liquor that ties whole antibody or its Fab that comprises immunoglobulin light and heavy chain that contains; Wherein, the expression of bait is optionally suppressed;

(13) measure the avidity for antigen from the non-antibody tying of supernatant liquor or its Fab, and identify the clone for example, with acceptable avidity (analyzing by Biacore);

(14) nucleotide sequence of the polynucleotide of mensuration coding heavily and in the evaluation of light chain immunoglobulin (Ig) clone.

Scope of the present invention also comprises for the identification of the polynucleotide of the heavy chain of encoding antibody and light chain immunoglobulin (Ig) or for the identification of the method that demonstrates the antibody of high stability.These class methods comprise the steps:

(a) for example, in eukaryotic host cell (pichia pastoris phaff) polynucleotide of coexpression bait and encode heavy and light chain are implemented denaturing agent to the antibody that comprises described chain simultaneously;

In one embodiment of the invention, denaturing agent with those of ordinary skills practitioner therefore by least part of sex change antibody of expection and suppress concentration or amount or magnitude (for example, in the sufficiently high temperature) existence of its ability of being combined with antigen.For example, possible denaturing agent comprises for example triton X-100(for example 1% or more of urea (for example 2,3,4,5 or 6 M or more), stain remover), dithiothreitol (DTT) (DTT) (for example 250 mM or 500 mM or more), Guanidinium hydrochloride, light (for example ultraviolet ray or visible ray), extreme pH(for example 1,2,3,14,13 or 12) or for example, for example, higher than for example 37 ℃ (42 ℃, 48 ℃ or 50 ℃) of temperature or its any combination (500 mM DTT/6 M urea) of approximately 4 ℃.

(b) identify the eukaryotic host cell of the bait of expression and Fc/ Fab (for example monovalent antibody fragments) dimerization, described fragment has detectable avidity (for example acceptable avidity) for antigen;

In one embodiment of the invention, also whether analysis package, containing the whole antibody being equal to the light and variable region of heavy chain of compound those of bait, has detectable avidity to measure them.

In one embodiment of the invention, whole antibody is secreted from host cell.In one embodiment of the invention, whole antibody is separated from host cell.

In one embodiment of the invention, suppress the expression of bait in host cell, but do not suppress the expression of whole antibody.In this embodiment of the present invention, host cell is only expressed whole antibody but is not expressed the bait with any remarkable quantity.In one embodiment of the invention, once the expression of bait is suppressed, just analyze the whole antibody being produced by host cell, to measure it, whether there is detectable avidity (for example acceptable avidity).

; With

(c) from cell, identify that described antibody or coding weigh and the polynucleotide of light chain, wherein one or more polynucleotide are optionally separated from host cell; Wherein under the existence of denaturing agent, demonstrate TPPA for the avidity of antigen for demonstrating high stability.In one embodiment of the invention, measure the nucleotide sequence of polynucleotide.

In one embodiment of the invention, for the people Fc immunoglobulin domains using at bait, comprise following aminoacid sequence:

（SEQ ID NO: 1）。

In one embodiment of the invention, SED1 comprises following aminoacid sequence:

（SEQ ID NO: 2）。

In one embodiment of the invention, for example merge, to the people Fc immunoglobulin (Ig) of SED1 polypeptide and signal sequence α mating factor signal sequence (MRFPSIFTAVLFAASSALA(SEQ ID NO:3) for example) be connected.

In one embodiment of the invention, comprise the bait merging to the people Fc immunoglobulin domains of SED1 polypeptide and comprise aminoacid sequence:

（SEQ ID NO: 4）。Fc immunoglobulin domains be have a underscore and connect with bold.SED1 polypeptide is after joint, and α mating factor signal peptide is before Fc.

Embodiment

The present invention expect illustration the present invention rather than restriction the present invention.Below disclosed method and composition (for example polypeptide, polynucleotide, plasmid, yeast cell) belongs to scope of the present invention.

embodiment 1:the structure of antibody display systems and purposes

antibody is shown the structure of bait

Following construction expression box.The polynucleotide that coding fixes on protein anchor the N-terminal of the cell surface anchorin matter on yeast cells wall are connected with the nucleotide sequence in encoding human IgG1 Fc district, and described cell surface anchorin matter contains fixed (GPI) posttranslational modification of the glycosyl-phosphatidyl inositol anchor adhering to inherently.The specific cell surface anchoring protein that we use is Saccharomyces Cerevisiae in S ed1 protein, it identifies by screening the cell walls of one group of plasmalemma protein matter, and described group has been used GPI protein prediction software (describing in international publication number WO09/111183) to identify.

In order to prepare the plasmid that contains bait box, the EcoRI forward PCR primer of the nucleotide sequence that use contains the yeast saccharomyces cerevisiae α mating factor signal sequence merging in the sequence upstream of coding IgG Fc N-terminal, with the SalI reverse primer of coding with the C-terminal of the IgG1 Fc of the sequence termination of coding GGGG joint, the optimized sequence of codon of synthetic human IgG1 Fc fragment.The plasmid that comprises anti-Her2 gene order is used as pcr template for EcoRI-α-mating factor signal sequence-Fc-GGGG-SalI fragment that increases.Use EcoRI and SalI endonuclease enzymic digestion PCR product and pGLY3033(to describe in international publication number WO09/111183).The EcoRI-SalI fragment of coding Fc is connected with EcoRI-SalI pGLY3033 main chain in frame, to generate plasmid pGLY9008(Fig. 2).This plasmid causes and can send at pichia pastoris phaff aOX1fc-SED1 box under the control of promoter sequence.As parental plasmid, it contains the pichia pastoris phaff that serves as the integrator locus in genome uRA6gene order and arsenite resistant gene, select containing on the substratum of Sodium metaarsenite allowing.

PGLY3033 plasmid sequence comprises nucleotide sequence:

（SEQ ID NO: 5）。

PGLY9008 plasmid sequence comprises nucleotide sequence:

（SEQ ID NO: 6）。

In order to test this configuration for show the ability of monovalent antibody fragments (comprising human IgG s) (1 heavy chain immunoglobulin and 1 light chain immunoglobulin (Ig) (H+L)) on yeast cells wall, pGLY9008 is introduced in GFI 5.0 bacterial strains, and described GFI 5.0 bacterial strains had previously been selected the expressive host as the anti-Her2 of people or anti-PCSK9 IgGs.Comprise sky bacterial strain (table 1) in contrast.

Table 1. yeast strain

Bacterial strain	mAb
		YGLY8316	Empty
YGLY18483	Anti-PCSK9(AX189)
		YGLY18281	Anti-PCSK9(AX132)
YGLY14755	Anti-PCSK9(1DG)
		YGLY13979	Anti-Her2
YGLY14836	Anti-Her2

* these Pichia Pastoris strains form part of the present invention.

The pichia pastoris phaff monoclonal antibody production bacterial strain of sugar transformation in table 1 is grown in 50 mL BMGY substratum, until the culture optical density(OD) at 600 nm places is 2 o'clock.By 1 M Sorbitol Powder washing three times for cell, and be resuspended in 1 mL 1 M Sorbitol Powder.The linearizing pGLY9008 of about 1-2 microgram SpeI is mixed with these competent cells.Use, for bore a hole manufacturers's program of pichia pastoris phaff internal specific of nov nucleic acid, is carried out and is transformed with BioRad electroporation apparatus.1 mL is reclaimed to substratum and add in cell, described cell is dull and stereotyped on yeast-soya peptone-dextrose (YSD) substratum upper berth with 50 μ g/mL arsenites subsequently.

fc-monovalent antibody fragments (H+L) is shown growth and the induction of yeast.The yeast two days of the sugar transformation of human IgG s and Fc-SED1 bait expression cassette is expressed in the 600 μ L BMGYs of use in 96 deep-well plates or 50 mL BMGY inoculations in 250 mL shaking flasks.By centrifugal collecting cell and abandoning supernatant.According to the method described in International Publication No. WO2007/061631, by the inducing cell that is incubated overnight in thering are 300 μ L of PMTi inhibitor or 25 mL BMMY.According to the scheme of manufacturers and albumin A, catch SDS-PAGE analysis, after induction, use κ ELISA to measure culture supernatant with regard to antibody expression.Data in Fig. 3 a and b are described respectively the result of these two mensuration.As summarized in Fig. 3, the whole antibody molecule of the secretion that the culture supernatant that discovery contains Fc-Sed1 protein bait contains similar level (compare with its parent strain (containing Fc-Sed1p) by 2 heavy chain immunoglobulins and 2 light chain immunoglobulin (Ig)s ((H2+L2)).The ability of the full IgG antibody of yeast secretary (H2+L2) is not disturbed in the existence of this indication Fc-Sed1p bait.

In order to measure the efficiency of the surface display antibody making in this way, cell is carried out to mark with the mouse anti human κ of APC 635 marks of the light chain of detection human antibody molecules, and process by flow cytometry.In brief, after the optical density(OD) 2 growing at 600 nm places, every kind of culture is washed by centrifugal formation agglomerate and in 100 μ L PBS.In 100 μ L phosphate buffered saline (PBS)s (PBS) of the mouse anti human κ light chain that cell is containing fluorescent mark (APC635) in room temperature (RT), incubation is 30 minutes, and washs in 100 μ L PBS.Before analyzing in flow cytometer, a hectolambda PBS is for resuspension agglomerate.

Use the cell of coexpression Fc-Sed1p bait and anti-Her2 or Fc-Sed1p bait and anti-PCSK9 to carry out flow cytometry.Preparation is wherein only expressed the empty bacterial strain of Fc-Sed1p bait or is expressed the contrast of the bacterial strain of the full length antibody (H2+L2) that does not contain Fc-Sed1p.The anti-κ that finds the bacterial strain displaying conspicuous level of the anti-Her2 of coexpression or anti-PCSK9 and Fc-Sed1p bait is combined, and lacks the bacterial strain demonstration background signal level of Fc-Sed1p bait.In Fig. 4 a-c, compare the fluorescence intensity from these experiments.Figure is presented at anti-Her2 and shows that cell and anti-PCSK9 show cell and these different fluorescence intensities that do not contain between the parent strain of Fc-Sed1p bait.It is worth mentioning that anti-Her2 shows that cell shows than the higher fluorescence intensity of anti-PCSK9 displaying cell.That consistent known with expression level about these two kinds of antibody of these results.

In order to determine that this method is for separating of the effectiveness of mixtures of antibodies, following execution shown the anti-PCSK9 monovalent antibody fragments of Fc-Sed1p (H+L) (bacterial strain YGLY21610) and the anti-Her2(H+L of Fc-Sed1p) fluorescence-activated cell sorting (FACS) of the cell mixture of (bacterial strain YGLY21614).To show anti-PCSK9(H+L) cell and show anti-Her2(H+L) cell with following ratio, mix: 1:0; 0:1; And 1:100.Cell carries out double-tagging with the streptavidin of the anti-human Fc Alexa 488 of goat and the biotinylated PCSK9 of 100 nM and APC 635 marks.Fig. 5 shows the anti-PCSK9(H+L of Fc-Sed1p/) can be in conjunction with biotinylated PCSK9, the anti-Her2(H+L of Fc-Sed1p/) can not.Two kinds of bacterial strains all with anti-human Fc Alexa 488 antibody responses.When the cell from two cultures, with the anti-PCSK9 of Fc-Sed1p, show 1:100 that cell (in circle) and the anti-Her2 of Fc-Sed1p show cell when mixing, two cell colonys that separate are visible.PCSK9 bonding agent number in this mixture with 1:100 than consistent, thereby make further to support this method to there is the soundness in the antibody of required antigen combination in screening.

Above-mentioned experiment confirms that Fc-Sed1p antibody display systems can be for showing IgG monovalent antibody fragments (H+L), and it retains the dimeric specific antigens combination of its corresponding whole antibody molecule (H2+L2).Next target is the novel antibody molecules that separation in this way and enrichment can be combined with any object antigen.For this reason, we utilize two libraries that recently build.Library one maintains (marinating) original sequence of light chain simultaneously and builds by changing the sequence of heavy chain of anti-PCSK9 antibody A X189.This library has the diversity of approximately 2500 unique sequences, and will be called as " BP550 ".Second library generates by maintaining original AX189 sequence of heavy chain and changing sequence of light chain.This library contains 4000 unique sequences of having an appointment, and will be called as " BP551 ".

BP550 and BP551 are transformed into sky 5.0 bacterial strains that bacterial strain YGLY21605(carries the pGLY9008 that expresses Fc-Sed1p as previously described) in, and dull and stereotyped on the YSD upper berth of containing 300 ug/ml bleomycin (zeocin).For each conversion, obtain about 50,000 bacterium colonies, thus provide in library likely the abundant statistics of sequence cover.From solid medium scraping, transform the bacterium colony that two libraries obtain, and separate inoculation is in the 250 mL shaking flasks that contain 50 mL YSG liquid nutrient mediums and 300 μ/mL bleomycin.By every kind of culture of 1 mL is inoculated in fresh selected liq substratum (YSG+ bleomycin), culture is gone down to posterity 3 times.Allowing to go down to posterity for the third time, it is saturated in YSG substratum, to grow to, and according to the method described in International Patent Publication No. WO 2007/061631, has PMTi inhibitor (PMTi4:L000001772; Concentration with 1 microgram/ml) in 25 mL BMMY, induce and spend the night.Comprise the anti-PCSK9(AX189 of bacterial strain YGLY21610(Fc-Sed1p)) and the anti-Her2 of YGLY21614(Fc-Sed1p) respectively as the positive and negative control.

After induction in 24 hours, four cultures grow to the optical density(OD) 2 at 600 nm places separately.Agglomerate is washed by centrifugal collection and with 100 μ L 1X PBS, subsequently mark in containing the 100 μ L PBS of anti-κ Alexa 488 and 100nM vitamin H-PCSK9.Mixture, room temperature incubation 30 minutes, is used 100 μ L PBS solution washings subsequently.Cell together with the streptavidin of room temperature and APC 635 marks in 100 μ L PBS incubation 10 minutes, and in PBS, wash 2X and submit to for FACS.

Use the flow cytometer point diagram being generated as border by YGLY21610 and YGLY21614 to carry out the potential bonding agent of gate, in FACS sorter, sorting is from 100 of the Liang Ge library BP550 of colony and BP551, the clone of 000 cell, and be collected in 5 mL YSG substratum.By within 5 days, allowing to reclaim culture in room temperature vibration.Sorting the 1st is taken turns storehouse and is inoculated in 50 mL YSG liquid nutrient mediums, and repeats identical process with induction and mark culture.To the 1st, take turns storehouse and carry out another and take turns sorting (the 2nd takes turns), and as above collecting cell and induction.In order to obtain single bacterium colony, 1000 cells of dull and stereotyped two two-wheeled sorting colonies (BP550 and BP551) on solid medium upper berth, and by κ ELISA and the affine ELISA sorting of PCSK9, to measure respectively protein titre and for the binding affinity of PCSK9.In addition, carry out yeast colony PCR amplified reaction, with the 2nd heavy chain and the light chain gene of taking turns clone that increase, it is submitted to for DNA sequence analysis.

As shown in Figure 6, use the two-wheeled sorting of biotinylated PCSK9 antigen to cause the significant enrichment of specificity PCSK9 bonding agent.The storehouse (Fig. 7) that PCSK9 ELISA relatively takes turns sorting for the library and the 2nd of BP550 and BP551 Pre-sorting.From the 2nd storehouse of taking turns sorting in two libraries, contain the bonding agent over the high per-cent of the colony of Pre-sorting.DNA sequencing confirms the enrichment for new anti-PCSK9 binding sequence.

The present invention is not limited by specific embodiments described herein in scope.In fact, scope of the present invention comprises the concrete embodiment of setting forth and herein not concrete other embodiments of setting forth herein; The concrete embodiment of setting forth not necessarily expects it is detailed herein.Except of the present invention a plurality of modifications of describing herein those will be apparent according to aforementioned specification for those skilled in the art.This type of modifies the scope that expection belongs to claim.

The application is referenced patents, patent application, publication, product description and scheme from start to finish, and its disclosure is incorporated herein by reference for all object integral body.

Sequence table

<110> Shaheen, Hussam H

Zha, Dongxing

<120>the FC-bait antibody display systems of surface anchoring

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tacaacccat ccactgacta cactactgac tacacagttg ttactgagta cactacttac 2040

tgtccagagc caactacttt cacaacaaac ggaaagactt acactgttac tgagcctact 2100

actttgacta tcactgactg tccatgtact atcgagaagc caactactac ttccactaca 2160

gagtatactg ttgttacaga atacacaaca tattgtcctg agccaacaac attcactact 2220

aatggaaaaa catacacagt tacagaacca actacattga caattacaga ttgtccttgt 2280

acaattgaga agtccgaggc tcctgaatct tctgttccag ttactgaatc caagggtact 2340

actactaaag aaactggtgt tactactaag cagactactg ctaacccatc cttgactgtt 2400

tccactgttg ttccagtttc ttcctctgct tcttcccact ccgttgttat caactccaac 2460

ggtgctaacg ttgttgttcc tggtgctttg ggattggctg gtgttgctat gttgttcttg 2520

taatagggcc ggccatttaa atacaggccc cttttccttt gtcgatatca tgtaattagt 2580

tatgtcacgc ttacattcac gccctcctcc cacatccgct ctaaccgaaa aggaaggagt 2640

tagacaacct gaagtctagg tccctattta ttttttttaa tagttatgtt agtattaaga 2700

acgttattta tatttcaaat ttttcttttt tttctgtaca aacgcgtgta cgcatgtaac 2760

attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag 2820

ctggatccgc ggccgcttac gcgccgttct tcgcttggtc ttgtatctcc ttacactgta 2880

tcttcccatt tgcgtttagg tggttatcaa aaactaaaag gaaaaatttc agatgtttat 2940

ctctaaggtt ttttcttttt acagtataac acgtgatgcg tcacgtggta ctagattacg 3000

taagttattt tggtccggtg ggtaagtggg taagaataga aagcatgaag gtttacaaaa 3060

acgcagtcac gaattattgc tacttcgagc ttggaaccac cccaaagatt atattgtact 3120

gatgcactac cttctcgatt ttgctcctcc aagaacctac gaaaaacatt tcttgagcct 3180

tttcaaccta gactacacat caagttattt aaggtatgtt ccgttaacat gtaagaaaag 3240

gagaggatag atcgtttatg gggtacgtcg cctgattcaa gcgtgaccat tcgaagaata 3300

ggccttcgaa agctgaataa agcaaatgtc agttgcgatt ggtatgctga caaattagca 3360

taaaaagcaa tagactttct aaccacctgt ttttttcctt ttactttatt tatattttgc 3420

caccgtacta acaagttcag acaaattaat taacaccatg tcagaagatc aaaaaagtga 3480

aaattccgta ccttctaagg ttaatatggt gaatcgcacc gatatactga ctacgatcaa 3540

gtcattgtca tggcttgact tgatgttgcc atttactata attctctcca taatcattgc 3600

agtaataatt tctgtctatg tgccttcttc ccgtcacact tttgacgctg aaggtcatcc 3660

caatctaatg ggagtgtcca ttcctttgac tgttggtatg attgtaatga tgattccccc 3720

gatctgcaaa gtttcctggg agtctattca caagtacttc tacaggagct atataaggaa 3780

gcaactagcc ctctcgttat ttttgaattg ggtcatcggt cctttgttga tgacagcatt 3840

ggcgtggatg gcgctattcg attataagga ataccgtcaa ggcattatta tgatcggagt 3900

agctagatgc attgccatgg tgctaatttg gaatcagatt gctggaggag acaatgatct 3960

ctgcgtcgtg cttgttatta caaactcgct tttacagatg gtattatatg caccattgca 4020

gatattttac tgttatgtta tttctcatga ccacctgaat acttcaaata gggtattatt 4080

cgaagaggtt gcaaagtctg tcggagtttt tctcggcata ccactgggaa ttggcattat 4140

catacgtttg ggaagtctta ccatagctgg taaaagtaat tatgaaaaat acattttgag 4200

atttatttct ccatgggcaa tgatcggatt tcattacact ttatttgtta tttttattag 4260

tagaggttat caatttatcc acgaaattgg ttctgcaata ttgtgctttg tcccattggt 4320

gctttacttc tttattgcat ggtttttgac cttcgcatta atgaggtact tatcaatatc 4380

taggagtgat acacaaagag aatgtagctg tgaccaagaa ctacttttaa agagggtctg 4440

gggaagaaag tcttgtgaag ctagcttttc tattacgatg acgcaatgtt tcactatggc 4500

ttcaaataat tttgaactat ccctggcaat tgctatttcc ttatatggta acaatagcaa 4560

gcaagcaata gctgcaacat ttgggccgtt gctagaagtt ccaattttat tgattttggc 4620

aatagtcgcg agaatcctta aaccatatta tatatggaac aatagaaatt aattaacagg 4680

ccccttttcc tttgtcgata tcatgtaatt agttatgtca cgcttacatt cacgccctcc 4740

tcccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat 4800

ttattttttt taatagttat gttagtatta agaacgttat ttatatttca aatttttctt 4860

ttttttctgt acaaacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag 4920

gttttgggac gctcgaaggc tttaatttgc aagctgcggc ctaaggcgcg ccaggccata 4980

atggcccaaa tgcaagagga cattagaaat gtgtttggta agaacatgaa gccggaggca 5040

tacaaacgat tcacagattt gaaggaggaa aacaaactgc atccaccgga agtgccagca 5100

gccgtgtatg ccaaccttgc tctcaaaggc attcctacgg atctgagtgg gaaatatctg 5160

agattcacag acccactatt ggaacagtac caaacctagt ttggccgatc catgattatg 5220

taatgcatat agtttttgtc gatgctcacc cgtttcgagt ctgtctcgta tcgtcttacg 5280

tataagttca agcatgttta ccaggtctgt tagaaactcc tttgtgaggg caggacctat 5340

tcgtctcggt cccgttgttt ctaagagact gtacagccaa gcgcagaatg gtggcattaa 5400

ccataagagg attctgatcg gacttggtct attggctatt ggaaccaccc tttacgggac 5460

aaccaaccct accaagactc ctattgcatt tgtggaacca gccacggaaa gagcgtttaa 5520

ggacggagac gtctctgtga tttttgttct cggaggtcca ggagctggaa aaggtaccca 5580

atgtgccaaa ctagtgagta attacggatt tgttcacctg tcagctggag acttgttacg 5640

tgcagaacag aagagggagg ggtctaagta tggagagatg atttcccagt atatcagaga 5700

tggactgata gtacctcaag aggtcaccat tgcgctcttg gagcaggcca tgaaggaaaa 5760

cttcgagaaa gggaagacac ggttcttgat tgatggattc cctcgtaaga tggaccaggc 5820

caaaactttt gaggaaaaag tcgcaaagtc caaggtgaca cttttctttg attgtcccga 5880

atcagtgctc cttgagagat tacttaaaag aggacagaca agcggaagag aggatgataa 5940

tgcggagagt atcaaaaaaa gattcaaaac attcgtggaa acttcgatgc ctgtggtgga 6000

ctatttcggg aagcaaggac gcgttttgaa ggtatcttgt gaccaccctg tggatcaagt 6060

gtattcacag gttgtgtcgg tgctaaaaga gaaggggatc tttgccgata acgagacgga 6120

gaataaataa acattgtaat aagatttaga ctgtgaatgt tctatgtaat atttttcgag 6180

atactgtatc tatctggtgt accgtatcac tctggacttg caaactcatt gattacttgt 6240

gcaatgggca agaaggatag ctctagaaag aagaagaaaa aggagccgcc tgaagagctg 6300

gatctttccg aggttgttcc aacttttggt tatgaggaat ttcatgttga gcaagaggag 6360

aatccggtcg atcaagacga acttgacggc cataatggcc tagcttggcg taatcatggt 6420

catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 6480

gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 6540

tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 6600

gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 6660

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 6720

tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 6780

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 6840

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6900

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6960

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 7020

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 7080

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 7140

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 7200

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 7260

ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 7320

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 7380

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 7440

acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 7500

tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 7560

agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 7620

gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 7680

agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 7740

cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 7800

ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 7860

cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 7920

cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 7980

ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 8040

tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 8100

catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 8160

gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 8220

gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 8280

tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 8340

catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 8400

aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 8460

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 8520

aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 8580

aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 8640

<210> 6

<211> 9180

<212> PRT

<213>artificial sequence

<220>

<223>plasmid pGLY9008

<400> 6

Thr Cys Gly Cys Gly Cys Gly Thr Thr Thr Cys Gly Gly Thr Gly Ala

1 5 10 15

Thr Gly Ala Cys Gly Gly Thr Gly Ala Ala Ala Ala Cys Cys Thr Cys

20 25 30

Thr Gly Ala Cys Ala Cys Ala Thr Gly Cys Ala Gly Cys Thr Cys Cys

35 40 45

Cys Gly Gly Ala Gly Ala Cys Gly Gly Thr Cys Ala Cys Ala Gly Cys

50 55 60

Thr Thr Gly Thr Cys Thr Gly Thr Ala Ala Gly Cys Gly Gly Ala Thr

65 70 75 80

Gly Cys Cys Gly Gly Gly Ala Gly Cys Ala Gly Ala Cys Ala Ala Gly

85 90 95

Cys Cys Cys Gly Thr Cys Ala Gly Gly Gly Cys Gly Cys Gly Thr Cys

100 105 110

Ala Gly Cys Gly Gly Gly Thr Gly Thr Thr Gly Gly Cys Gly Gly Gly

115 120 125

Thr Gly Thr Cys Gly Gly Gly Gly Cys Thr Gly Gly Cys Thr Thr Ala

130 135 140

Ala Cys Thr Ala Thr Gly Cys Gly Gly Cys Ala Thr Cys Ala Gly Ala

145 150 155 160

Gly Cys Ala Gly Ala Thr Thr Gly Thr Ala Cys Thr Gly Ala Gly Ala

165 170 175

Gly Thr Gly Cys Ala Cys Cys Ala Thr Ala Thr Gly Cys Gly Gly Thr

180 185 190

Gly Thr Gly Ala Ala Ala Thr Ala Cys Cys Gly Cys Ala Cys Ala Gly

195 200 205

Ala Thr Gly Cys Gly Thr Ala Ala Gly Gly Ala Gly Ala Ala Ala Ala

210 215 220

Thr Ala Cys Cys Gly Cys Ala Thr Cys Ala Gly Gly Cys Gly Cys Cys

225 230 235 240

Ala Thr Thr Cys Gly Cys Cys Ala Thr Thr Cys Ala Gly Gly Cys Thr

245 250 255

Gly Cys Gly Cys Ala Ala Cys Thr Gly Thr Thr Gly Gly Gly Ala Ala

260 265 270

Gly Gly Gly Cys Gly Ala Thr Cys Gly Gly Thr Gly Cys Gly Gly Gly

275 280 285

Cys Cys Thr Cys Thr Thr Cys Gly Cys Thr Ala Thr Thr Ala Cys Gly

290 295 300

Cys Cys Ala Gly Cys Thr Gly Gly Cys Gly Ala Ala Ala Gly Gly Gly

305 310 315 320

Gly Gly Ala Thr Gly Thr Gly Cys Thr Gly Cys Ala Ala Gly Gly Cys

325 330 335

Gly Ala Thr Thr Ala Ala Gly Thr Thr Gly Gly Gly Thr Ala Ala Cys

340 345 350

Gly Cys Cys Ala Gly Gly Gly Thr Thr Thr Thr Cys Cys Cys Ala Gly

355 360 365

Thr Cys Ala Cys Gly Ala Cys Gly Thr Thr Gly Thr Ala Ala Ala Ala

370 375 380

Cys Gly Ala Cys Gly Gly Cys Cys Ala Gly Thr Gly Ala Ala Thr Thr

385 390 395 400

Gly Ala Gly Ala Thr Cys Thr Ala Ala Cys Ala Thr Cys Cys Ala Ala

405 410 415

Ala Gly Ala Cys Gly Ala Ala Ala Gly Gly Thr Thr Gly Ala Ala Thr

420 425 430

Gly Ala Ala Ala Cys Cys Thr Thr Thr Thr Thr Gly Cys Cys Ala Thr

435 440 445

Cys Cys Gly Ala Cys Ala Thr Cys Cys Ala Cys Ala Gly Gly Thr Cys

450 455 460

Cys Ala Thr Thr Cys Thr Cys Ala Cys Ala Cys Ala Thr Ala Ala Gly

465 470 475 480

Thr Gly Cys Cys Ala Ala Ala Cys Gly Cys Ala Ala Cys Ala Gly Gly

485 490 495

Ala Gly Gly Gly Gly Ala Thr Ala Cys Ala Cys Thr Ala Gly Cys Ala

500 505 510

Gly Cys Ala Gly Ala Cys Cys Gly Thr Thr Gly Cys Ala Ala Ala Cys

515 520 525

Gly Cys Ala Gly Gly Ala Cys Cys Thr Cys Cys Ala Cys Thr Cys Cys

530 535 540

Thr Cys Thr Thr Cys Thr Cys Cys Thr Cys Ala Ala Cys Ala Cys Cys

545 550 555 560

Cys Ala Cys Thr Thr Thr Thr Gly Cys Cys Ala Thr Cys Gly Ala Ala

565 570 575

Ala Ala Ala Cys Cys Ala Gly Cys Cys Cys Ala Gly Thr Thr Ala Thr

580 585 590

Thr Gly Gly Gly Cys Thr Thr Gly Ala Thr Thr Gly Gly Ala Gly Cys

595 600 605

Thr Cys Gly Cys Thr Cys Ala Thr Thr Cys Cys Ala Ala Thr Thr Cys

610 615 620

Cys Thr Thr Cys Thr Ala Thr Thr Ala Gly Gly Cys Thr Ala Cys Thr

625 630 635 640

Ala Ala Cys Ala Cys Cys Ala Thr Gly Ala Cys Thr Thr Thr Ala Thr

645 650 655

Thr Ala Gly Cys Cys Thr Gly Thr Cys Thr Ala Thr Cys Cys Thr Gly

660 665 670

Gly Cys Cys Cys Cys Cys Cys Thr Gly Gly Cys Gly Ala Gly Gly Thr

675 680 685

Thr Cys Ala Thr Gly Thr Thr Thr Gly Thr Thr Thr Ala Thr Thr Thr

690 695 700

Cys Cys Gly Ala Ala Thr Gly Cys Ala Ala Cys Ala Ala Gly Cys Thr

705 710 715 720

Cys Cys Gly Cys Ala Thr Thr Ala Cys Ala Cys Cys Cys Gly Ala Ala

725 730 735

Cys Ala Thr Cys Ala Cys Thr Cys Cys Ala Gly Ala Thr Gly Ala Gly

740 745 750

Gly Gly Cys Thr Thr Thr Cys Thr Gly Ala Gly Thr Gly Thr Gly Gly

755 760 765

Gly Gly Thr Cys Ala Ala Ala Thr Ala Gly Thr Thr Thr Cys Ala Thr

770 775 780

Gly Thr Thr Cys Cys Cys Cys Ala Ala Ala Thr Gly Gly Cys Cys Cys

785 790 795 800

Ala Ala Ala Ala Cys Thr Gly Ala Cys Ala Gly Thr Thr Thr Ala Ala

805 810 815

Ala Cys Gly Cys Thr Gly Thr Cys Thr Thr Gly Gly Ala Ala Cys Cys

820 825 830

Thr Ala Ala Thr Ala Thr Gly Ala Cys Ala Ala Ala Ala Gly Cys Gly

835 840 845

Thr Gly Ala Thr Cys Thr Cys Ala Thr Cys Cys Ala Ala Gly Ala Thr

850 855 860

Gly Ala Ala Cys Thr Ala Ala Gly Thr Thr Thr Gly Gly Thr Thr Cys

865 870 875 880

Gly Thr Thr Gly Ala Ala Ala Thr Gly Cys Thr Ala Ala Cys Gly Gly

885 890 895

Cys Cys Ala Gly Thr Thr Gly Gly Thr Cys Ala Ala Ala Ala Ala Gly

900 905 910

Ala Ala Ala Cys Thr Thr Cys Cys Ala Ala Ala Ala Gly Thr Cys Gly

915 920 925

Gly Cys Ala Thr Ala Cys Cys Gly Thr Thr Thr Gly Thr Cys Thr Thr

930 935 940

Gly Thr Thr Thr Gly Gly Thr Ala Thr Thr Gly Ala Thr Thr Gly Ala

945 950 955 960

Cys Gly Ala Ala Thr Gly Cys Thr Cys Ala Ala Ala Ala Ala Thr Ala

965 970 975

Ala Thr Cys Thr Cys Ala Thr Thr Ala Ala Thr Gly Cys Thr Thr Ala

980 985 990

Gly Cys Gly Cys Ala Gly Thr Cys Thr Cys Thr Cys Thr Ala Thr Cys

995 1000 1005

Gly Cys Thr Thr Cys Thr Gly Ala Ala Cys Cys Cys Cys Gly Gly

1010 1015 1020

Thr Gly Cys Ala Cys Cys Thr Gly Thr Gly Cys Cys Gly Ala Ala

1025 1030 1035

Ala Cys Gly Cys Ala Ala Ala Thr Gly Gly Gly Gly Ala Ala Ala

1040 1045 1050

Cys Ala Cys Cys Cys Gly Cys Thr Thr Thr Thr Thr Gly Gly Ala

1055 1060 1065

Thr Gly Ala Thr Thr Ala Thr Gly Cys Ala Thr Thr Gly Thr Cys

1070 1075 1080

Thr Cys Cys Ala Cys Ala Thr Thr Gly Thr Ala Thr Gly Cys Thr

1085 1090 1095

Thr Cys Cys Ala Ala Gly Ala Thr Thr Cys Thr Gly Gly Thr Gly

1100 1105 1110

Gly Gly Ala Ala Thr Ala Cys Thr Gly Cys Thr Gly Ala Thr Ala

1115 1120 1125

Gly Cys Cys Thr Ala Ala Cys Gly Thr Thr Cys Ala Thr Gly Ala

1130 1135 1140

Thr Cys Ala Ala Ala Ala Thr Thr Thr Ala Ala Cys Thr Gly Thr

1145 1150 1155

Thr Cys Thr Ala Ala Cys Cys Cys Cys Thr Ala Cys Thr Thr Gly

1160 1165 1170

Ala Cys Ala Gly Cys Ala Ala Thr Ala Thr Ala Thr Ala Ala Ala

1175 1180 1185

Cys Ala Gly Ala Ala Gly Gly Ala Ala Gly Cys Thr Gly Cys Cys

1190 1195 1200

Cys Thr Gly Thr Cys Thr Thr Ala Ala Ala Cys Cys Thr Thr Thr

1205 1210 1215

Thr Thr Thr Thr Thr Thr Ala Thr Cys Ala Thr Cys Ala Thr Thr

1220 1225 1230

Ala Thr Thr Ala Gly Cys Thr Thr Ala Cys Thr Thr Thr Cys Ala

1235 1240 1245

Thr Ala Ala Thr Thr Gly Cys Gly Ala Cys Thr Gly Gly Thr Thr

1250 1255 1260

Cys Cys Ala Ala Thr Thr Gly Ala Cys Ala Ala Gly Cys Thr Thr

1265 1270 1275

Thr Thr Gly Ala Thr Thr Thr Thr Ala Ala Cys Gly Ala Cys Thr

1280 1285 1290

Thr Thr Thr Ala Ala Cys Gly Ala Cys Ala Ala Cys Thr Thr Gly

1295 1300 1305

Ala Gly Ala Ala Gly Ala Thr Cys Ala Ala Ala Ala Ala Ala Cys

1310 1315 1320

Ala Ala Cys Thr Ala Ala Thr Thr Ala Thr Thr Cys Gly Ala Ala

1325 1330 1335

Ala Cys Gly Gly Ala Ala Thr Thr Cys Ala Cys Gly Ala Thr Gly

1340 1345 1350

Ala Gly Ala Thr Thr Thr Cys Cys Thr Thr Cys Ala Ala Thr Thr

1355 1360 1365

Thr Thr Thr Ala Cys Thr Gly Cys Thr Gly Thr Thr Thr Thr Ala

1370 1375 1380

Thr Thr Cys Gly Cys Ala Gly Cys Ala Thr Cys Cys Thr Cys Cys

1385 1390 1395

Gly Cys Ala Thr Thr Ala Gly Cys Thr Gly Ala Cys Ala Ala Gly

1400 1405 1410

Ala Cys Ala Cys Ala Thr Ala Cys Thr Thr Gly Thr Cys Cys Ala

1415 1420 1425

Cys Cys Ala Thr Gly Thr Cys Cys Ala Gly Cys Thr Cys Cys Ala

1430 1435 1440

Gly Ala Ala Thr Thr Gly Thr Thr Gly Gly Gly Thr Gly Gly Thr

1445 1450 1455

Cys Cys Ala Thr Cys Cys Gly Thr Thr Thr Thr Cys Thr Thr Gly

1460 1465 1470

Thr Thr Cys Cys Cys Ala Cys Cys Ala Ala Ala Gly Cys Cys Ala

1475 1480 1485

Ala Ala Gly Gly Ala Cys Ala Cys Thr Thr Thr Gly Ala Thr Gly

1490 1495 1500

Ala Thr Cys Thr Cys Cys Ala Gly Ala Ala Cys Thr Cys Cys Ala

1505 1510 1515

Gly Ala Gly Gly Thr Thr Ala Cys Ala Thr Gly Thr Gly Thr Thr

1520 1525 1530

Gly Thr Thr Gly Thr Thr Gly Ala Cys Gly Thr Thr Thr Cys Thr

1535 1540 1545

Cys Ala Cys Gly Ala Gly Gly Ala Cys Cys Cys Ala Gly Ala Gly

1550 1555 1560

Gly Thr Thr Ala Ala Gly Thr Thr Cys Ala Ala Cys Thr Gly Gly

1565 1570 1575

Thr Ala Cys Gly Thr Thr Gly Ala Cys Gly Gly Thr Gly Thr Thr

1580 1585 1590

Gly Ala Ala Gly Thr Thr Cys Ala Cys Ala Ala Cys Gly Cys Thr

1595 1600 1605

Ala Ala Gly Ala Cys Thr Ala Ala Gly Cys Cys Ala Ala Gly Ala

1610 1615 1620

Gly Ala Ala Gly Ala Gly Cys Ala Gly Thr Ala Cys Ala Ala Cys

1625 1630 1635

Thr Cys Cys Ala Cys Thr Thr Ala Cys Ala Gly Ala Gly Thr Thr

1640 1645 1650

Gly Thr Thr Thr Cys Cys Gly Thr Thr Thr Thr Gly Ala Cys Thr

1655 1660 1665

Gly Thr Thr Thr Thr Gly Cys Ala Cys Cys Ala Gly Gly Ala Cys

1670 1675 1680

Thr Gly Gly Thr Thr Gly Ala Ala Cys Gly Gly Thr Ala Ala Ala

1685 1690 1695

Gly Ala Ala Thr Ala Cys Ala Ala Gly Thr Gly Thr Ala Ala Gly

1700 1705 1710

Gly Thr Thr Thr Cys Cys Ala Ala Cys Ala Ala Gly Gly Cys Thr

1715 1720 1725

Thr Thr Gly Cys Cys Ala Gly Cys Thr Cys Cys Ala Ala Thr Cys

1730 1735 1740

Gly Ala Ala Ala Ala Gly Ala Cys Thr Ala Thr Cys Thr Cys Cys

1745 1750 1755

Ala Ala Gly Gly Cys Thr Ala Ala Gly Gly Gly Thr Cys Ala Ala

1760 1765 1770

Cys Cys Ala Ala Gly Ala Gly Ala Gly Cys Cys Ala Cys Ala Gly

1775 1780 1785

Gly Thr Thr Thr Ala Cys Ala Cys Thr Thr Thr Gly Cys Cys Ala

1790 1795 1800

Cys Cys Ala Thr Cys Cys Ala Gly Ala Gly Ala Ala Gly Ala Gly

1805 1810 1815

Ala Thr Gly Ala Cys Thr Ala Ala Gly Ala Ala Cys Cys Ala Gly

1820 1825 1830

Gly Thr Thr Thr Cys Cys Thr Thr Gly Ala Cys Thr Thr Gly Thr

1835 1840 1845

Thr Thr Gly Gly Thr Thr Ala Ala Ala Gly Gly Ala Thr Thr Cys

1850 1855 1860

Thr Ala Cys Cys Cys Ala Thr Cys Cys Gly Ala Cys Ala Thr Thr

1865 1870 1875

Gly Cys Thr Gly Thr Thr Gly Ala Gly Thr Gly Gly Gly Ala Ala

1880 1885 1890

Thr Cys Thr Ala Ala Cys Gly Gly Thr Cys Ala Ala Cys Cys Ala

1895 1900 1905

Gly Ala Gly Ala Ala Cys Ala Ala Cys Thr Ala Cys Ala Ala Gly

1910 1915 1920

Ala Cys Thr Ala Cys Thr Cys Cys Ala Cys Cys Ala Gly Thr Thr

1925 1930 1935

Thr Thr Gly Gly Ala Thr Thr Cys Thr Gly Ala Thr Gly Gly Thr

1940 1945 1950

Thr Cys Cys Thr Thr Cys Thr Thr Cys Thr Thr Gly Thr Ala Cys

1955 1960 1965

Thr Cys Cys Ala Ala Gly Thr Thr Gly Ala Cys Thr Gly Thr Thr

1970 1975 1980

Gly Ala Cys Ala Ala Gly Thr Cys Cys Ala Gly Ala Thr Gly Gly

1985 1990 1995

Cys Ala Ala Cys Ala Gly Gly Gly Thr Ala Ala Cys Gly Thr Thr

2000 2005 2010

Thr Thr Cys Thr Cys Cys Thr Gly Thr Thr Cys Cys Gly Thr Thr

2015 2020 2025

Ala Thr Gly Cys Ala Thr Gly Ala Gly Gly Cys Thr Thr Thr Gly

2030 2035 2040

Cys Ala Cys Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Cys Thr

2045 2050 2055

Cys Ala Ala Ala Ala Gly Thr Cys Cys Thr Thr Gly Thr Cys Thr

2060 2065 2070

Thr Thr Gly Thr Cys Cys Cys Cys Thr Gly Gly Thr Gly Gly Thr

2075 2080 2085

Gly Gly Thr Gly Gly Thr Gly Thr Cys Gly Ala Cys Cys Ala Ala

2090 2095 2100

Thr Thr Cys Thr Cys Thr Ala Ala Cys Thr Cys Thr Ala Cys Thr

2105 2110 2115

Thr Cys Cys Gly Cys Thr Thr Cys Cys Thr Cys Thr Ala Cys Thr

2120 2125 2130

Gly Ala Cys Gly Thr Thr Ala Cys Thr Thr Cys Cys Thr Cys Cys

2135 2140 2145

Thr Cys Cys Thr Cys Thr Ala Thr Thr Thr Cys Thr Ala Cys Thr

2150 2155 2160

Thr Cys Cys Thr Cys Cys Gly Gly Thr Thr Cys Cys Gly Thr Thr

2165 2170 2175

Ala Cys Thr Ala Thr Thr Ala Cys Thr Thr Cys Cys Thr Cys Thr

2180 2185 2190

Gly Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Ala Thr Cys Thr

2195 2200 2205

Gly Ala Cys Ala Ala Cys Gly Gly Thr Ala Cys Thr Thr Cys Thr

2210 2215 2220

Ala Cys Thr Gly Cys Thr Gly Cys Thr Cys Cys Ala Ala Cys Thr

2225 2230 2235

Gly Ala Ala Ala Cys Thr Thr Cys Thr Ala Cys Thr Gly Ala Gly

2240 2245 2250

Gly Cys Thr Cys Cys Thr Ala Cys Thr Ala Cys Thr Gly Cys Thr

2255 2260 2265

Ala Thr Thr Cys Cys Ala Ala Cys Thr Ala Ala Cys Gly Gly Ala

2270 2275 2280

Ala Cys Thr Thr Cys Cys Ala Cys Ala Gly Ala Gly Gly Cys Thr

2285 2290 2295

Cys Cys Ala Ala Cys Ala Ala Cys Ala Gly Cys Thr Ala Thr Cys

2300 2305 2310

Cys Cys Thr Ala Cys Ala Ala Ala Cys Gly Gly Thr Ala Cys Ala

2315 2320 2325

Thr Cys Cys Ala Cys Thr Gly Ala Ala Gly Cys Thr Cys Cys Thr

2330 2335 2340

Ala Cys Thr Gly Ala Cys Ala Cys Thr Ala Cys Thr Ala Cys Ala

2345 2350 2355

Gly Ala Ala Gly Cys Thr Cys Cys Ala Ala Cys Thr Ala Cys Thr

2360 2365 2370

Gly Cys Thr Thr Thr Gly Cys Cys Thr Ala Cys Thr Ala Ala Thr

2375 2380 2385

Gly Gly Thr Ala Cys Ala Thr Cys Ala Ala Cys Ala Gly Ala Gly

2390 2395 2400

Gly Cys Thr Cys Cys Thr Ala Cys Ala Gly Ala Thr Ala Cys Ala

2405 2410 2415

Ala Cys Ala Ala Cys Thr Gly Ala Ala Gly Cys Thr Cys Cys Ala

2420 2425 2430

Ala Cys Ala Ala Cys Thr Gly Gly Ala Thr Thr Gly Cys Cys Ala

2435 2440 2445

Ala Cys Ala Ala Ala Cys Gly Gly Thr Ala Cys Thr Ala Cys Thr

2450 2455 2460

Thr Cys Thr Gly Cys Thr Thr Thr Cys Cys Cys Ala Cys Cys Ala

2465 2470 2475

Ala Cys Thr Ala Cys Thr Thr Cys Cys Thr Thr Gly Cys Cys Ala

2480 2485 2490

Cys Cys Ala Thr Cys Cys Ala Ala Cys Ala Cys Thr Ala Cys Thr

2495 2500 2505

Ala Cys Thr Ala Cys Thr Cys Cys Ala Cys Cys Ala Thr Ala Cys

2510 2515 2520

Ala Ala Cys Cys Cys Ala Thr Cys Cys Ala Cys Thr Gly Ala Cys

2525 2530 2535

Thr Ala Cys Ala Cys Thr Ala Cys Thr Gly Ala Cys Thr Ala Cys

2540 2545 2550

Ala Cys Ala Gly Thr Thr Gly Thr Thr Ala Cys Thr Gly Ala Gly

2555 2560 2565

Thr Ala Cys Ala Cys Thr Ala Cys Thr Thr Ala Cys Thr Gly Thr

2570 2575 2580

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Gly Gly Cys Thr Cys Cys Gly Cys Cys Cys Cys Cys Cys Thr Gly

7370 7375 7380

Ala Cys Gly Ala Gly Cys Ala Thr Cys Ala Cys Ala Ala Ala Ala

7385 7390 7395

Ala Thr Cys Gly Ala Cys Gly Cys Thr Cys Ala Ala Gly Thr Cys

7400 7405 7410

Ala Gly Ala Gly Gly Thr Gly Gly Cys Gly Ala Ala Ala Cys Cys

7415 7420 7425

Cys Gly Ala Cys Ala Gly Gly Ala Cys Thr Ala Thr Ala Ala Ala

7430 7435 7440

Gly Ala Thr Ala Cys Cys Ala Gly Gly Cys Gly Thr Thr Thr Cys

7445 7450 7455

Cys Cys Cys Cys Thr Gly Gly Ala Ala Gly Cys Thr Cys Cys Cys

7460 7465 7470

Thr Cys Gly Thr Gly Cys Gly Cys Thr Cys Thr Cys Cys Thr Gly

7475 7480 7485

Thr Thr Cys Cys Gly Ala Cys Cys Cys Thr Gly Cys Cys Gly Cys

7490 7495 7500

Thr Thr Ala Cys Cys Gly Gly Ala Thr Ala Cys Cys Thr Gly Thr

7505 7510 7515

Cys Cys Gly Cys Cys Thr Thr Thr Cys Thr Cys Cys Cys Thr Thr

7520 7525 7530

Cys Gly Gly Gly Ala Ala Gly Cys Gly Thr Gly Gly Cys Gly Cys

7535 7540 7545

Thr Thr Thr Cys Thr Cys Ala Thr Ala Gly Cys Thr Cys Ala Cys

7550 7555 7560

Gly Cys Thr Gly Thr Ala Gly Gly Thr Ala Thr Cys Thr Cys Ala

7565 7570 7575

Gly Thr Thr Cys Gly Gly Thr Gly Thr Ala Gly Gly Thr Cys Gly

7580 7585 7590

Thr Thr Cys Gly Cys Thr Cys Cys Ala Ala Gly Cys Thr Gly Gly

7595 7600 7605

Gly Cys Thr Gly Thr Gly Thr Gly Cys Ala Cys Gly Ala Ala Cys

7610 7615 7620

Cys Cys Cys Cys Cys Gly Thr Thr Cys Ala Gly Cys Cys Cys Gly

7625 7630 7635

Ala Cys Cys Gly Cys Thr Gly Cys Gly Cys Cys Thr Thr Ala Thr

7640 7645 7650

Cys Cys Gly Gly Thr Ala Ala Cys Thr Ala Thr Cys Gly Thr Cys

7655 7660 7665

Thr Thr Gly Ala Gly Thr Cys Cys Ala Ala Cys Cys Cys Gly Gly

7670 7675 7680

Thr Ala Ala Gly Ala Cys Ala Cys Gly Ala Cys Thr Thr Ala Thr

7685 7690 7695

Cys Gly Cys Cys Ala Cys Thr Gly Gly Cys Ala Gly Cys Ala Gly

7700 7705 7710

Cys Cys Ala Cys Thr Gly Gly Thr Ala Ala Cys Ala Gly Gly Ala

7715 7720 7725

Thr Thr Ala Gly Cys Ala Gly Ala Gly Cys Gly Ala Gly Gly Thr

7730 7735 7740

Ala Thr Gly Thr Ala Gly Gly Cys Gly Gly Thr Gly Cys Thr Ala

7745 7750 7755

Cys Ala Gly Ala Gly Thr Thr Cys Thr Thr Gly Ala Ala Gly Thr

7760 7765 7770

Gly Gly Thr Gly Gly Cys Cys Thr Ala Ala Cys Thr Ala Cys Gly

7775 7780 7785

Gly Cys Thr Ala Cys Ala Cys Thr Ala Gly Ala Ala Gly Gly Ala

7790 7795 7800

Cys Ala Gly Thr Ala Thr Thr Thr Gly Gly Thr Ala Thr Cys Thr

7805 7810 7815

Gly Cys Gly Cys Thr Cys Thr Gly Cys Thr Gly Ala Ala Gly Cys

7820 7825 7830

Cys Ala Gly Thr Thr Ala Cys Cys Thr Thr Cys Gly Gly Ala Ala

7835 7840 7845

Ala Ala Ala Gly Ala Gly Thr Thr Gly Gly Thr Ala Gly Cys Thr

7850 7855 7860

Cys Thr Thr Gly Ala Thr Cys Cys Gly Gly Cys Ala Ala Ala Cys

7865 7870 7875

Ala Ala Ala Cys Cys Ala Cys Cys Gly Cys Thr Gly Gly Thr Ala

7880 7885 7890

Gly Cys Gly Gly Thr Gly Gly Thr Thr Thr Thr Thr Thr Thr Gly

7895 7900 7905

Thr Thr Thr Gly Cys Ala Ala Gly Cys Ala Gly Cys Ala Gly Ala

7910 7915 7920

Thr Thr Ala Cys Gly Cys Gly Cys Ala Gly Ala Ala Ala Ala Ala

7925 7930 7935

Ala Ala Gly Gly Ala Thr Cys Thr Cys Ala Ala Gly Ala Ala Gly

7940 7945 7950

Ala Thr Cys Cys Thr Thr Thr Gly Ala Thr Cys Thr Thr Thr Thr

7955 7960 7965

Cys Thr Ala Cys Gly Gly Gly Gly Thr Cys Thr Gly Ala Cys Gly

7970 7975 7980

Cys Thr Cys Ala Gly Thr Gly Gly Ala Ala Cys Gly Ala Ala Ala

7985 7990 7995

Ala Cys Thr Cys Ala Cys Gly Thr Thr Ala Ala Gly Gly Gly Ala

8000 8005 8010

Thr Thr Thr Thr Gly Gly Thr Cys Ala Thr Gly Ala Gly Ala Thr

8015 8020 8025

Thr Ala Thr Cys Ala Ala Ala Ala Ala Gly Gly Ala Thr Cys Thr

8030 8035 8040

Thr Cys Ala Cys Cys Thr Ala Gly Ala Thr Cys Cys Thr Thr Thr

8045 8050 8055

Thr Ala Ala Ala Thr Thr Ala Ala Ala Ala Ala Thr Gly Ala Ala

8060 8065 8070

Gly Thr Thr Thr Thr Ala Ala Ala Thr Cys Ala Ala Thr Cys Thr

8075 8080 8085

Ala Ala Ala Gly Thr Ala Thr Ala Thr Ala Thr Gly Ala Gly Thr

8090 8095 8100

Ala Ala Ala Cys Thr Thr Gly Gly Thr Cys Thr Gly Ala Cys Ala

8105 8110 8115

Gly Thr Thr Ala Cys Cys Ala Ala Thr Gly Cys Thr Thr Ala Ala

8120 8125 8130

Thr Cys Ala Gly Thr Gly Ala Gly Gly Cys Ala Cys Cys Thr Ala

8135 8140 8145

Thr Cys Thr Cys Ala Gly Cys Gly Ala Thr Cys Thr Gly Thr Cys

8150 8155 8160

Thr Ala Thr Thr Thr Cys Gly Thr Thr Cys Ala Thr Cys Cys Ala

8165 8170 8175

Thr Ala Gly Thr Thr Gly Cys Cys Thr Gly Ala Cys Thr Cys Cys

8180 8185 8190

Cys Cys Gly Thr Cys Gly Thr Gly Thr Ala Gly Ala Thr Ala Ala

8195 8200 8205

Cys Thr Ala Cys Gly Ala Thr Ala Cys Gly Gly Gly Ala Gly Gly

8210 8215 8220

Gly Cys Thr Thr Ala Cys Cys Ala Thr Cys Thr Gly Gly Cys Cys

8225 8230 8235

Cys Cys Ala Gly Thr Gly Cys Thr Gly Cys Ala Ala Thr Gly Ala

8240 8245 8250

Thr Ala Cys Cys Gly Cys Gly Ala Gly Ala Cys Cys Cys Ala Cys

8255 8260 8265

Gly Cys Thr Cys Ala Cys Cys Gly Gly Cys Thr Cys Cys Ala Gly

8270 8275 8280

Ala Thr Thr Thr Ala Thr Cys Ala Gly Cys Ala Ala Thr Ala Ala

8285 8290 8295

Ala Cys Cys Ala Gly Cys Cys Ala Gly Cys Cys Gly Gly Ala Ala

8300 8305 8310

Gly Gly Gly Cys Cys Gly Ala Gly Cys Gly Cys Ala Gly Ala Ala

8315 8320 8325

Gly Thr Gly Gly Thr Cys Cys Thr Gly Cys Ala Ala Cys Thr Thr

8330 8335 8340

Thr Ala Thr Cys Cys Gly Cys Cys Thr Cys Cys Ala Thr Cys Cys

8345 8350 8355

Ala Gly Thr Cys Thr Ala Thr Thr Ala Ala Thr Thr Gly Thr Thr

8360 8365 8370

Gly Cys Cys Gly Gly Gly Ala Ala Gly Cys Thr Ala Gly Ala Gly

8375 8380 8385

Thr Ala Ala Gly Thr Ala Gly Thr Thr Cys Gly Cys Cys Ala Gly

8390 8395 8400

Thr Thr Ala Ala Thr Ala Gly Thr Thr Thr Gly Cys Gly Cys Ala

8405 8410 8415

Ala Cys Gly Thr Thr Gly Thr Thr Gly Cys Cys Ala Thr Thr Gly

8420 8425 8430

Cys Thr Ala Cys Ala Gly Gly Cys Ala Thr Cys Gly Thr Gly Gly

8435 8440 8445

Thr Gly Thr Cys Ala Cys Gly Cys Thr Cys Gly Thr Cys Gly Thr

8450 8455 8460

Thr Thr Gly Gly Thr Ala Thr Gly Gly Cys Thr Thr Cys Ala Thr

8465 8470 8475

Thr Cys Ala Gly Cys Thr Cys Cys Gly Gly Thr Thr Cys Cys Cys

8480 8485 8490

Ala Ala Cys Gly Ala Thr Cys Ala Ala Gly Gly Cys Gly Ala Gly

8495 8500 8505

Thr Thr Ala Cys Ala Thr Gly Ala Thr Cys Cys Cys Cys Cys Ala

8510 8515 8520

Thr Gly Thr Thr Gly Thr Gly Cys Ala Ala Ala Ala Ala Ala Gly

8525 8530 8535

Cys Gly Gly Thr Thr Ala Gly Cys Thr Cys Cys Thr Thr Cys Gly

8540 8545 8550

Gly Thr Cys Cys Thr Cys Cys Gly Ala Thr Cys Gly Thr Thr Gly

8555 8560 8565

Thr Cys Ala Gly Ala Ala Gly Thr Ala Ala Gly Thr Thr Gly Gly

8570 8575 8580

Cys Cys Gly Cys Ala Gly Thr Gly Thr Thr Ala Thr Cys Ala Cys

8585 8590 8595

Thr Cys Ala Thr Gly Gly Thr Thr Ala Thr Gly Gly Cys Ala Gly

8600 8605 8610

Cys Ala Cys Thr Gly Cys Ala Thr Ala Ala Thr Thr Cys Thr Cys

8615 8620 8625

Thr Thr Ala Cys Thr Gly Thr Cys Ala Thr Gly Cys Cys Ala Thr

8630 8635 8640

Cys Cys Gly Thr Ala Ala Gly Ala Thr Gly Cys Thr Thr Thr Thr

8645 8650 8655

Cys Thr Gly Thr Gly Ala Cys Thr Gly Gly Thr Gly Ala Gly Thr

8660 8665 8670

Ala Cys Thr Cys Ala Ala Cys Cys Ala Ala Gly Thr Cys Ala Thr

8675 8680 8685

Thr Cys Thr Gly Ala Gly Ala Ala Thr Ala Gly Thr Gly Thr Ala

8690 8695 8700

Thr Gly Cys Gly Gly Cys Gly Ala Cys Cys Gly Ala Gly Thr Thr

8705 8710 8715

Gly Cys Thr Cys Thr Thr Gly Cys Cys Cys Gly Gly Cys Gly Thr

8720 8725 8730

Cys Ala Ala Thr Ala Cys Gly Gly Gly Ala Thr Ala Ala Thr Ala

8735 8740 8745

Cys Cys Gly Cys Gly Cys Cys Ala Cys Ala Thr Ala Gly Cys Ala

8750 8755 8760

Gly Ala Ala Cys Thr Thr Thr Ala Ala Ala Ala Gly Thr Gly Cys

8765 8770 8775

Thr Cys Ala Thr Cys Ala Thr Thr Gly Gly Ala Ala Ala Ala Cys

8780 8785 8790

Gly Thr Thr Cys Thr Thr Cys Gly Gly Gly Gly Cys Gly Ala Ala

8795 8800 8805

Ala Ala Cys Thr Cys Thr Cys Ala Ala Gly Gly Ala Thr Cys Thr

8810 8815 8820

Thr Ala Cys Cys Gly Cys Thr Gly Thr Thr Gly Ala Gly Ala Thr

8825 8830 8835

Cys Cys Ala Gly Thr Thr Cys Gly Ala Thr Gly Thr Ala Ala Cys

8840 8845 8850

Cys Cys Ala Cys Thr Cys Gly Thr Gly Cys Ala Cys Cys Cys Ala

8855 8860 8865

Ala Cys Thr Gly Ala Thr Cys Thr Thr Cys Ala Gly Cys Ala Thr

8870 8875 8880

Cys Thr Thr Thr Thr Ala Cys Thr Thr Thr Cys Ala Cys Cys Ala

8885 8890 8895

Gly Cys Gly Thr Thr Thr Cys Thr Gly Gly Gly Thr Gly Ala Gly

8900 8905 8910

Cys Ala Ala Ala Ala Ala Cys Ala Gly Gly Ala Ala Gly Gly Cys

8915 8920 8925

Ala Ala Ala Ala Thr Gly Cys Cys Gly Cys Ala Ala Ala Ala Ala

8930 8935 8940

Ala Gly Gly Gly Ala Ala Thr Ala Ala Gly Gly Gly Cys Gly Ala

8945 8950 8955

Cys Ala Cys Gly Gly Ala Ala Ala Thr Gly Thr Thr Gly Ala Ala

8960 8965 8970

Thr Ala Cys Thr Cys Ala Thr Ala Cys Thr Cys Thr Thr Cys Cys

8975 8980 8985

Thr Thr Thr Thr Thr Cys Ala Ala Thr Ala Thr Thr Ala Thr Thr

8990 8995 9000

Gly Ala Ala Gly Cys Ala Thr Thr Thr Ala Thr Cys Ala Gly Gly

9005 9010 9015

Gly Thr Thr Ala Thr Thr Gly Thr Cys Thr Cys Ala Thr Gly Ala

9020 9025 9030

Gly Cys Gly Gly Ala Thr Ala Cys Ala Thr Ala Thr Thr Thr Gly

9035 9040 9045

Ala Ala Thr Gly Thr Ala Thr Thr Thr Ala Gly Ala Ala Ala Ala

9050 9055 9060

Ala Thr Ala Ala Ala Cys Ala Ala Ala Thr Ala Gly Gly Gly Gly

9065 9070 9075

Thr Thr Cys Cys Gly Cys Gly Cys Ala Cys Ala Thr Thr Thr Cys

9080 9085 9090

Cys Cys Cys Gly Ala Ala Ala Ala Gly Thr Gly Cys Cys Ala Cys

9095 9100 9105

Cys Thr Gly Ala Cys Gly Thr Cys Thr Ala Ala Gly Ala Ala Ala

9110 9115 9120

Cys Cys Ala Thr Thr Ala Thr Thr Ala Thr Cys Ala Thr Gly Ala

9125 9130 9135

Cys Ala Thr Thr Ala Ala Cys Cys Thr Ala Thr Ala Ala Ala Ala

9140 9145 9150

Ala Thr Ala Gly Gly Cys Gly Thr Ala Thr Cys Ala Cys Gly Ala

9155 9160 9165

Gly Gly Cys Cys Cys Thr Thr Thr Cys Gly Thr Cys

9170 9175 9180

Claims

1. an antibody display systems, it comprises:

(a) separated host cell;

(b) comprise the bait merging to the heavy Fc immunoglobulin domains of surface anchoring polypeptide;

(c) one or more polynucleotide of coding immunoglobulin light chain variable region; With

(d) one or more polynucleotide of coding immunoglobulin heavy chain variable region.

2. the antibody display systems of claim 1, it also comprises

(i) the non-whole antibody tying that comprises described immunoglobulin light and heavy chain; And/or

(ii) the monovalent antibody fragments compound with the Fc part of described bait.

3. the antibody display systems of any one in claim 1-2, wherein said host cell is Pichia cell.

4. the antibody display systems of any one in claim 1-3,

The different colony in the heredity from immunoglobulin light chain variable region of described one or more polynucleotide of immunoglobulin light chain variable region of wherein encoding; And/or,

The different colony in the heredity from immunoglobulin heavy chain variable region of described one or more polynucleotide of immunoglobulin heavy chain variable region of wherein encoding.

5. the antibody display systems of any one in claim 1-4, the polynucleotide that wherein said host cell comprises the bait of encoding, described polynucleotide are operationally combined with adjustable promotor.

6. a separated bait polypeptide, it comprises optionally and merges Fc immunoglobulin domains or its function fragment to surface anchoring polypeptide or its function fragment by peptide linker; The optional N-terminal of described bait polypeptide merges to signal peptide.

7. the polypeptide of claim 6, wherein said surface anchoring polypeptide is SED-1 or its function fragment.

8. the polypeptide of any one in claim 6-7, wherein said Fc immunoglobulin domains is IgG1, IgG2, IgG3 or IgG4 immunoglobulin (Ig).

9. separated polynucleotide, the polypeptide of any one in its coding claim 6-8.

10. a carrier, the polynucleotide that it comprises claim 9.

11. 1 kinds of separated host cells, it comprises the polypeptide of any one or the polynucleotide of coding said polypeptide in claim 6-8.

The host cell of 12. claims 11, it also comprises one or more polynucleotide of the immunoglobulin light chain variable region of encoding; And/or one or more polynucleotide of coding immunoglobulin heavy chain variable region.

The host cell of any one in 13. claim 11-12, it is Pichia cell.

The host cell of any one in 14. claim 11-13, wherein said polypeptide is positioned on described surface of cell membrane.

15. 1 kinds of compositions that comprise the host cell of any one in claim 11-14, it also comprises the non-whole antibody tying that comprises described immunoglobulin light and heavy chain; And/or with the Fc/ Fab of the compound antibody of the Fc part of described bait; Optionally, wherein said whole antibody or Fc/ Fab and object antigen are compound.

16. 1 kinds of methods of whether being combined with antigen-specific for measuring antibody or its Fab, it comprises makes antibody display systems contact with described antigen; Wherein said antibody display systems comprises:

(a) separated eukaryotic host cell, the polynucleotide that it comprises the light chain immunoglobulin of encoding; Polynucleotide with coding heavy chain immunoglobulin; With

(b) comprise and merge to the lip-deep surface anchoring polypeptide of described eukaryotic host cell or the Fc immunoglobulin domains of its function fragment or the bait of its function fragment;

On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab heavy and light chain immunoglobulin compound;

Whether and measuring described Fc/ Fab is combined with described antigen-specific;

If wherein described monovalent antibody fragments is combined with described antigen-specific, described TPPA is antigen described in specific binding.

17. 1 kinds for the identification of following method:

(i) the antibody of being combined with antigen-specific or its Fab; Or

(ii) the polynucleotide of the light chain immunoglobulin of the polynucleotide of the heavy chain immunoglobulin of encoding said antibody or fragment and/or encoding said antibody or fragment;

It comprises makes antibody display systems contact with described antigen, and wherein said antibody display systems comprises:

If wherein observe the described specific binding with described antigen, identify described antibody or fragment or polynucleotide.

The method of any one in 18. claim 16-17, it also comprises the separated polynucleotide of identifying.

The method of any one in 19. claim 16-18, it also comprises the expression that suppresses described bait, measures subsequently the antibody of described evaluation or its Fab for the avidity of described antigen.

The method of any one in 20. claim 16-19, it also comprises the recombinant expressed immunoglobulin chain by described polynucleotide encoding, the antibody or its Fab that comprise described immunoglobulin (Ig) with optionally separating, and the optional pharmaceutical preparation that comprises the described antibody of combination or its Fab and pharmaceutically acceptable carrier that produces.

21. 1 kinds for the preparation of the method that comprises following antibody display systems:

(a) separated eukaryotic host cell;

(b) comprise the bait merging to the people Fc immunoglobulin domains of surface anchoring polypeptide;

(c) one or more polynucleotide of coding immunoglobulin light chain variable region;

(d) one or more polynucleotide of coding immunoglobulin heavy chain variable region;

Described method comprises the polynucleotide of the described bait of coding, described one or more polynucleotide of coding immunoglobulin light chain variable region; Introduce in described eukaryotic host cell with described one or more polynucleotide of coding immunoglobulin heavy chain variable region.

22. 1 kinds of methods for the preparation of antibody or its Fab, it comprises one or more polynucleotide of coding immunoglobulin light chain variable region; And/or one or more polynucleotide of coding immunoglobulin heavy chain variable region introduce in the separated eukaryotic host cell that comprises bait, described bait comprises people Fc immunoglobulin domains or its function fragment merging to surface anchoring polypeptide or its function fragment; With cultivate described host cell expressing thus under the polynucleotide of coding immunoglobulin chain and the condition by described chain formation antibody or its Fab; Wherein said bait is operationally combined with adjustable promotor, and when described immunoglobulin chain is expressed, bait is expressed suppressed.

23. 1 kinds of methods for the preparation of antibody or its Fab, it is included in and allows under the light chain immunoglobulin of described antibody or fragment and condition that heavy chain immunoglobulin is expressed the eukaryotic host cell of culture of isolated in growth medium; Wherein said eukaryotic host cell comprises:

(i) the encode polynucleotide of described light chain immunoglobulin; Polynucleotide with the described heavy chain immunoglobulin of encoding said antibody or fragment; With

(ii) comprise and merge to the lip-deep surface anchoring polypeptide of described eukaryotic host cell or the Fc immunoglobulin domains of its function fragment or the bait of its function fragment;

On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab heavy and light chain immunoglobulin compound; And wherein the expression of bait is optionally repressed; Wherein said antibody or fragment are optionally secreted by described eukaryotic host cell; Described method optionally comprises from described eukaryotic host cell and separated described antibody or fragment substratum.

24. 1 kinds for measuring the method for sugar to the effect of the antibody of being combined with antigen-specific or its Fab, and it comprises makes antibody display systems be combined with described antigen; Wherein said antibody display systems comprises:

(a) separated eucaryon is controlled glycosylated host cell, the polynucleotide that it comprises the light chain immunoglobulin of encoding; Polynucleotide with coding heavy chain immunoglobulin; With

(b) comprise surface anchoring polypeptide or the Fc immunoglobulin domains of its function fragment or the bait of its function fragment merging on described host cell surface;

On the Fc of wherein said bait and host cell surface, comprise described immunoglobulin (Ig) Fc/ Fab heavy and light chain immunoglobulin compound; Wherein said heavy or light chain comprises described sugar;

Whether measure described Fc/ Fab is combined with described antigen-specific;

The antibody that mensuration comprises described sugar or its Fab are for the avidity of antigen; With

The antibody that other aspects of the avidity of more described antibody or its Fab and the described sugar of shortage are equal to or the avidity of its Fab;

If wherein comprise the antibody of described sugar or the avidity of its Fab higher than lacking the antibody of described sugar or the avidity of its Fab, described sugar determination is for increasing the avidity for antigen, if and/or wherein comprise the antibody of described sugar or the avidity of its Fab lower than lacking the antibody of described sugar or the avidity of its Fab, described sugar determination is for reducing the avidity for antigen.